{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T22:38:06Z","timestamp":1778711886661,"version":"3.51.4"},"reference-count":52,"publisher":"Public Library of Science (PLoS)","issue":"5","license":[{"start":{"date-parts":[[2021,5,28]],"date-time":"2021-05-28T00:00:00Z","timestamp":1622160000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004063","name":"Knut och Alice Wallenbergs Stiftelse","doi-asserted-by":"publisher","award":["KAW2017.0022"],"award-info":[{"award-number":["KAW2017.0022"]}],"id":[{"id":"10.13039\/501100004063","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.<\/jats:p>","DOI":"10.1371\/journal.pone.0252624","type":"journal-article","created":{"date-parts":[[2021,5,28]],"date-time":"2021-05-28T18:53:38Z","timestamp":1622228018000},"page":"e0252624","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":6,"title":["Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity"],"prefix":"10.1371","volume":"16","author":[{"given":"Zhirong","family":"Fu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Srinivas","family":"Akula","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chang","family":"Qiao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinhye","family":"Ryu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9601-3057","authenticated-orcid":true,"given":"Gurdeep","family":"Chahal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lawrence","family":"de Garavilla","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jukka","family":"Kervinen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Thorpe","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1459-3815","authenticated-orcid":true,"given":"Lars","family":"Hellman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2021,5,28]]},"reference":[{"issue":"17","key":"pone.0252624.ref001","doi-asserted-by":"crossref","first-page":"6232","DOI":"10.1073\/pnas.85.17.6232","article-title":"Mouse lysozyme M gene: isolation, characterization, and expression studies","volume":"85","author":"M Cross","year":"1988","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"pone.0252624.ref002","first-page":"1","article-title":"Genomic organization and evolution of ruminant lysozyme c genes","volume":"36","author":"DM Irwin","year":"2015","journal-title":"Dongwuxue Yanjiu"},{"issue":"6","key":"pone.0252624.ref003","first-page":"1082","article-title":"Evolution of cow nonstomach lysozyme genes. Genome\/National Research Council Canada = Genome\/Conseil national de recherches","volume":"47","author":"DM Irwin","year":"2004","journal-title":"Canada"},{"issue":"1\u20132","key":"pone.0252624.ref004","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/0378-1119(96)00352-6","article-title":"Structural deviations in a bovine low expression lysozyme-encoding gene active in tissues other than stomach","volume":"178","author":"M Henke","year":"1996","journal-title":"Gene"},{"issue":"2","key":"pone.0252624.ref005","doi-asserted-by":"crossref","first-page":"684","DOI":"10.3168\/jds.2010-3217","article-title":"Changes of protein content and its fractions in bovine milk from different breeds subject to somatic cell count","volume":"94","author":"Z Litwinczuk","year":"2011","journal-title":"J Dairy Sci"},{"issue":"6","key":"pone.0252624.ref006","doi-asserted-by":"crossref","first-page":"544","DOI":"10.1111\/pai.12265","article-title":"Anti-infective proteins in breast milk and asthma-associated phenotypes during early childhood","volume":"25","author":"G Zhang","year":"2014","journal-title":"Pediatr Allergy Immunol"},{"issue":"4","key":"pone.0252624.ref007","first-page":"347","article-title":"Lysozyme in Livestock: A Guide to Selection for Disease Resistance: a Review","volume":"2","author":"NR Sahoo","year":"2012","journal-title":"J Anim Sci Adv"},{"issue":"7","key":"pone.0252624.ref008","doi-asserted-by":"crossref","first-page":"2174","DOI":"10.1002\/jsfa.8026","article-title":"Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression","volume":"97","author":"J Cieslak","year":"2017","journal-title":"J Sci Food Agric"},{"issue":"2","key":"pone.0252624.ref009","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1016\/j.biochi.2007.10.009","article-title":"Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions","volume":"90","author":"B Korkmaz","year":"2008","journal-title":"Biochimie"},{"issue":"24","key":"pone.0252624.ref010","doi-asserted-by":"crossref","first-page":"4981","DOI":"10.1182\/blood-2010-01-257287","article-title":"Mast cell proteases: multifaceted regulators of inflammatory disease","volume":"115","author":"G Pejler","year":"2010","journal-title":"Blood"},{"key":"pone.0252624.ref011","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1007\/978-1-4419-9533-9_12","article-title":"Mast cell proteases as protective and inflammatory mediators","volume":"716","author":"GH Caughey","year":"2011","journal-title":"Adv Exp Med Biol"},{"issue":"1","key":"pone.0252624.ref012","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1515\/hsz-2013-0211","article-title":"Granule proteases of hematopoietic cells, a family of versatile inflammatory mediators\u2014an update on their cleavage specificity, in vivo substrates, and evolution","volume":"395","author":"L Hellman","year":"2014","journal-title":"Biol Chem"},{"issue":"11","key":"pone.0252624.ref013","doi-asserted-by":"crossref","first-page":"e0143091","DOI":"10.1371\/journal.pone.0143091","article-title":"Granule Associated Serine Proteases of Hematopoietic Cells\u2014An Analysis of Their Appearance and Diversification during Vertebrate Evolution","volume":"10","author":"S Akula","year":"2015","journal-title":"PLoS One"},{"issue":"3","key":"pone.0252624.ref014","first-page":"866","article-title":"Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Purification and properties. European journal of biochemistry\/","volume":"227","author":"TS Zamolodchikova","year":"1995","journal-title":"FEBS"},{"issue":"2","key":"pone.0252624.ref015","first-page":"612","article-title":"Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase. European journal of biochemistry\/","volume":"249","author":"TS Zamolodchikova","year":"1997","journal-title":"FEBS"},{"issue":"Pt 3","key":"pone.0252624.ref016","doi-asserted-by":"crossref","first-page":"665","DOI":"10.1042\/bj3210665","article-title":"Sheep mast cell proteinase-1: characterization as a member of a new class of dual-specific ruminant chymases","volume":"321","author":"AD Pemberton","year":"1997","journal-title":"Biochem J"},{"issue":"Pt 3","key":"pone.0252624.ref017","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1042\/bj3330801","article-title":"Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity","volume":"333","author":"SM McAleese","year":"1998","journal-title":"Biochem J"},{"issue":"2\u20133","key":"pone.0252624.ref018","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/S0014-5793(00)01092-9","article-title":"Activation of recombinant proenteropeptidase by duodenase","volume":"466","author":"TS Zamolodchikova","year":"2000","journal-title":"FEBS letters"},{"issue":"4","key":"pone.0252624.ref019","first-page":"1171","article-title":"Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts. European journal of biochemistry\/","volume":"269","author":"AD Pemberton","year":"2002","journal-title":"FEBS"},{"issue":"6","key":"pone.0252624.ref020","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1046\/j.1365-2222.2000.00831.x","article-title":"cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract","volume":"30","author":"AD Pemberton","year":"2000","journal-title":"Clin Exp Allergy"},{"issue":"1","key":"pone.0252624.ref021","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1093\/intimm\/dxn128","article-title":"The extended substrate specificity of the human mast cell chymase reveals a serine protease with well-defined substrate recognition profile","volume":"21","author":"MK Andersson","year":"2009","journal-title":"Int Immunol"},{"issue":"4","key":"pone.0252624.ref022","doi-asserted-by":"crossref","first-page":"e0195077","DOI":"10.1371\/journal.pone.0195077","article-title":"Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities","volume":"13","author":"M Thorpe","year":"2018","journal-title":"PLoS One"},{"issue":"43","key":"pone.0252624.ref023","doi-asserted-by":"crossref","first-page":"30468","DOI":"10.1074\/jbc.274.43.30468","article-title":"The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes","volume":"274","author":"KM Edwards","year":"1999","journal-title":"J Biol Chem"},{"issue":"9","key":"pone.0252624.ref024","doi-asserted-by":"crossref","first-page":"5360","DOI":"10.4049\/jimmunol.1002292","article-title":"How immune peptidases change specificity: cathepsin G gained tryptic function but lost efficiency during primate evolution","volume":"185","author":"WW Raymond","year":"2010","journal-title":"J Immunol"},{"issue":"4","key":"pone.0252624.ref025","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1083\/jcb.200606073","article-title":"The major human and mouse granzymes are structurally and functionally divergent","volume":"175","author":"D Kaiserman","year":"2006","journal-title":"The Journal of cell biology"},{"issue":"5","key":"pone.0252624.ref026","doi-asserted-by":"crossref","first-page":"e0154886","DOI":"10.1371\/journal.pone.0154886","article-title":"Asp-ase Activity of the Opossum Granzyme B Supports the Role of Granzyme B as Part of Anti-Viral Immunity Already during Early Mammalian Evolution","volume":"11","author":"Z Fu","year":"2016","journal-title":"PLoS One"},{"issue":"7","key":"pone.0252624.ref027","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1016\/j.bcp.2010.06.014","article-title":"Potency variation of small-molecule chymase inhibitors across species","volume":"80","author":"J Kervinen","year":"2010","journal-title":"Biochem Pharmacol"},{"key":"pone.0252624.ref028","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1016\/j.dci.2018.11.019","article-title":"Extended cleavage specificity of sheep mast cell protease-2: A classical chymase with preference to aromatic P1 substrate residues","volume":"92","author":"Z Fu","year":"2019","journal-title":"Developmental and comparative immunology"},{"issue":"2","key":"pone.0252624.ref029","doi-asserted-by":"crossref","first-page":"e31756","DOI":"10.1371\/journal.pone.0031756","article-title":"The extended cleavage specificity of human thrombin","volume":"7","author":"M Gallwitz","year":"2012","journal-title":"PLoS One"},{"issue":"6","key":"pone.0252624.ref030","doi-asserted-by":"crossref","first-page":"e0129511","DOI":"10.1371\/journal.pone.0129511","article-title":"The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin","volume":"10","author":"G Chahal","year":"2015","journal-title":"PLoS One"},{"issue":"6","key":"pone.0252624.ref031","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1093\/intimm\/dxq021","article-title":"The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase","volume":"22","author":"M Gallwitz","year":"2010","journal-title":"Int Immunol"},{"issue":"10","key":"pone.0252624.ref032","doi-asserted-by":"crossref","first-page":"2255","DOI":"10.1111\/j.1742-4658.2010.07642.x","article-title":"Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2\u2019 of substrates","volume":"277","author":"MK Andersson","year":"2010","journal-title":"Febs J"},{"key":"pone.0252624.ref033","doi-asserted-by":"crossref","first-page":"771","DOI":"10.1093\/intimm\/dxs081","article-title":"Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis): an interesting animal model for the analysis of the function of the human mast cell chymase","volume":"12","author":"M Thorpe","year":"2012","journal-title":"Int Immunol"},{"issue":"6","key":"pone.0252624.ref034","doi-asserted-by":"crossref","first-page":"e0131720","DOI":"10.1371\/journal.pone.0131720","article-title":"rMCP-2, the Major Rat Mucosal Mast Cell Protease, an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins","volume":"10","author":"Z Fu","year":"2015","journal-title":"PLoS One"},{"key":"pone.0252624.ref035","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.dci.2016.05.013","article-title":"Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells","volume":"63","author":"M Thorpe","year":"2016","journal-title":"Developmental and comparative immunology"},{"issue":"8","key":"pone.0252624.ref036","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1134\/S0006297913080130","article-title":"Expression of duodenase-like protein in epitheliocytes of Brunner\u2019s glands in human duodenal mucosa","volume":"78","author":"TS Zamolodchikova","year":"2013","journal-title":"Biochemistry Biokhimiia"},{"issue":"1 Pt 1","key":"pone.0252624.ref037","first-page":"G19","article-title":"Secretion of colonic isozyme of lysozyme in association with cecotrophy of rabbits","volume":"247","author":"VM Camara","year":"1984","journal-title":"Am J Physiol"},{"issue":"1","key":"pone.0252624.ref038","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1006\/mpev.1993.1007","article-title":"Evolution of rodent lysozymes: isolation and sequence of the rat lysozyme genes","volume":"2","author":"TC Yeh","year":"1993","journal-title":"Mol Phylogenet Evol"},{"issue":"18","key":"pone.0252624.ref039","doi-asserted-by":"crossref","first-page":"11607","DOI":"10.1016\/S0021-9258(18)90907-5","article-title":"Stomach lysozymes of ruminants. I. Distribution and catalytic properties","volume":"259","author":"DE Dobson","year":"1984","journal-title":"J Biol Chem"},{"issue":"2","key":"pone.0252624.ref040","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1006\/mpev.1996.0025","article-title":"Evolution of stomach lysozyme: the pig lysozyme gene","volume":"5","author":"M Yu","year":"1996","journal-title":"Mol Phylogenet Evol"},{"issue":"6146","key":"pone.0252624.ref041","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1038\/330401a0","article-title":"Adaptive evolution in the stomach lysozymes of foregut fermenters","volume":"330","author":"CB Stewart","year":"1987","journal-title":"Nature"},{"issue":"6","key":"pone.0252624.ref042","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1007\/BF02338801","article-title":"Molecular genetics and evolution of stomach and nonstomach lysozymes in the hoatzin","volume":"42","author":"JR Kornegay","year":"1996","journal-title":"J Mol Evol"},{"key":"pone.0252624.ref043","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1186\/1471-2148-11-166","article-title":"Evolution of the mammalian lysozyme gene family","volume":"11","author":"DM Irwin","year":"2011","journal-title":"BMC Evol Biol"},{"issue":"1","key":"pone.0252624.ref044","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1007\/s12038-010-0015-5","article-title":"Lysozymes in the animal kingdom","volume":"35","author":"L Callewaert","year":"2010","journal-title":"J Biosci"},{"issue":"2","key":"pone.0252624.ref045","doi-asserted-by":"crossref","first-page":"123","DOI":"10.3168\/jds.S0022-0302(49)92019-6","article-title":"Some physical and chemical properties of bovone saliva which may affect rumen digestion and synthesis","volume":"32","author":"JT Reid","year":"1949","journal-title":"Journal of Dairy Science"},{"issue":"1","key":"pone.0252624.ref046","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/0003-2697(87)90021-2","article-title":"Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction","volume":"162","author":"P Chomczynski","year":"1987","journal-title":"Anal Biochem"},{"issue":"3","key":"pone.0252624.ref047","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1093\/sysbio\/sys029","article-title":"MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space","volume":"61","author":"F Ronquist","year":"2012","journal-title":"Syst Biol"},{"issue":"8","key":"pone.0252624.ref048","doi-asserted-by":"crossref","first-page":"875","DOI":"10.1096\/fj.01-0879fje","article-title":"Generation of therapeutic antibody responses against IgE through vaccination","volume":"16","author":"M Vernersson","year":"2002","journal-title":"Faseb J"},{"issue":"6","key":"pone.0252624.ref049","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1002\/elps.1150090603","article-title":"Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250","volume":"9","author":"V Neuhoff","year":"1988","journal-title":"Electrophoresis"},{"issue":"21","key":"pone.0252624.ref050","doi-asserted-by":"crossref","first-page":"18579","DOI":"10.1074\/jbc.M110356200","article-title":"Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile","volume":"277","author":"U Karlson","year":"2002","journal-title":"J Biol Chem"},{"issue":"41","key":"pone.0252624.ref051","doi-asserted-by":"crossref","first-page":"39625","DOI":"10.1074\/jbc.M301512200","article-title":"Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity","volume":"278","author":"U Karlson","year":"2003","journal-title":"J Biol Chem"},{"issue":"3","key":"pone.0252624.ref052","doi-asserted-by":"crossref","first-page":"766","DOI":"10.1016\/j.molimm.2007.06.360","article-title":"The extended cleavage specificity of the rodent beta-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase","volume":"45","author":"MK Andersson","year":"2008","journal-title":"Mol Immunol"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0252624","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,11,4]],"date-time":"2023-11-04T08:33:14Z","timestamp":1699086794000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0252624"}},"subtitle":[],"editor":[{"given":"Israel","family":"Silman","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2021,5,28]]},"references-count":52,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2021,5,28]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0252624","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,28]]}}}