{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T18:05:10Z","timestamp":1789495510006,"version":"build-2803163510"},"reference-count":42,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T00:00:00Z","timestamp":1789430400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Variants in the\n                    <jats:italic>SPINT2<\/jats:italic>\n                    gene, encoding HAI-2, cause the syndromic form of autosomal recessive Congenital Sodium Diarrhea (CSD), a life-threatening disease with early-onset persistent diarrhea and stools containing elevated levels of sodium. A newly discovered variant, HAI-2 Y68C, causes a milder non-syndromic CSD-like condition in an isolated case. The present investigation compares biochemical features of the HAI-2 Y68C with two newly discovered syndromic CSD-causing variants, HAI-2 Y129C and R148H, and two previously characterized syndromic CSD-causing variants, HAI-2 F161V and G168S. HAI-2 contains two Kunitz domains that inhibit a range of serine proteases, including matriptase and prostasin. HAI-2 is co-located with target proteases mainly in the ER and the early secretory pathway but also on the apical plasma membrane of polarized cells. All investigated variants of HAI-2 were expressed with a similar steady-state protein level and weak pericellular staining as wild-type HAI-2. However, none of the CSD-causing variants were capable of inhibiting prostasin as efficiently as wild-type HAI-2. In addition, the HAI-2 Y68C variant was unable to inhibit matriptase, constituting a clear biochemical difference between the single variant causing non-syndromic CSD and the variants causing syndromic CSD. The HAI-2 Y68C variant may affect the phenotype by altering matriptase catalyzed activation of prostasin in the early secretory pathway. We hypothesize that CSD, whether syndromic or non-syndromic, is caused by a lack of inhibitory activity of HAI-2 in the early secretory pathway and\/or on the apical plasma membrane, resulting in uncontrolled proteolytic activity leading to proteolytic modification of sodium transporters\/channels like NHE3 and ENaC. NHE3 and ENaC are normally located on the apical plasma membrane and are mainly responsible for the uptake of sodium from the gut lumen. Oral administration of a protease inhibitor mimicking HAI-2 wild-type could potentially help to reduce the degradation of proteins important for sodium uptake across the apical plasma membrane.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0358119","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T17:36:56Z","timestamp":1789493816000},"page":"e0358119","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["A biochemical difference between HAI-2 variants causing syndromic and non-syndromic Congenital Sodium Diarrhea"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-3668-4746","authenticated-orcid":true,"given":"Maiken Jaller","family":"Jensen","sequence":"first","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0478-2295","authenticated-orcid":true,"given":"Monika","family":"Mrackova","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annika Weile","family":"Nonboe","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-7909-5927","authenticated-orcid":true,"given":"Emilie Tina","family":"Lembke","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine","family":"Schar","sequence":"additional","affiliation":[{"name":"Aarhus University","place":["Aarhus, Denmark"],"department":["Department of Molecular Biology and Genetics"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jan Kristian","family":"Jensen","sequence":"additional","affiliation":[{"name":"Aarhus University","place":["Aarhus, Denmark"],"department":["Department of Molecular Biology and Genetics"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6181-2705","authenticated-orcid":true,"given":"Asli","family":"Silahtaroglu","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ren\u00e9","family":"Villadsen","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9920-0982","authenticated-orcid":true,"given":"Lotte Katrine","family":"Vogel","sequence":"additional","affiliation":[{"name":"University of Copenhagen","place":["Copenhagen N, Denmark"],"department":["Department of Cellular and Molecular Medicine"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,15]]},"reference":[{"issue":"2","key":"pone.0358119.ref001","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1097\/MPG.0000000000001139","article-title":"Congenital Sodium Diarrhea","volume":"63","author":"AR Janecke","year":"2016","journal-title":"Journal of Pediatric Gastroenterology and Nutrition"},{"issue":"2","key":"pone.0358119.ref002","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.ajhg.2009.01.004","article-title":"Mutations in SPINT2 cause a syndromic form of congenital sodium diarrhea","volume":"84","author":"P Heinz-Erian","year":"2009","journal-title":"Am J Hum Genet"},{"issue":"23","key":"pone.0358119.ref003","doi-asserted-by":"crossref","first-page":"6614","DOI":"10.1093\/hmg\/ddv367","article-title":"Reduced sodium\/proton exchanger NHE3 activity causes congenital sodium diarrhea","volume":"24","author":"AR Janecke","year":"2015","journal-title":"Hum Mol Genet"},{"issue":"8","key":"pone.0358119.ref004","doi-asserted-by":"crossref","first-page":"1306","DOI":"10.1136\/gutjnl-2015-309441","article-title":"Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C","volume":"65","author":"T M\u00fcller","year":"2016","journal-title":"Gut"},{"issue":"18","key":"pone.0358119.ref005","doi-asserted-by":"crossref","first-page":"2835","DOI":"10.1016\/j.febslet.2012.07.028","article-title":"The multiple and enigmatic roles of guanylyl cyclase C in intestinal homeostasis","volume":"586","author":"N Arshad","year":"2012","journal-title":"FEBS Lett"},{"key":"pone.0358119.ref006","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1242\/jeb.028605","article-title":"NHE3 regulatory complexes","volume":"212","author":"M Donowitz","year":"2009","journal-title":"J Exp Biol"},{"issue":"3","key":"pone.0358119.ref007","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1007\/s00439-013-1380-6","article-title":"Genetic characterization of congenital tufting enteropathy: epcam associated phenotype and involvement of SPINT2 in the syndromic form","volume":"133","author":"J Salomon","year":"2014","journal-title":"Hum Genet"},{"issue":"4","key":"pone.0358119.ref008","doi-asserted-by":"crossref","first-page":"997","DOI":"10.1002\/ajmg.a.38637","article-title":"Congenital sodium diarrhea and chorioretinal coloboma with optic disc coloboma in a patient with biallelic SPINT2 mutations, including p.(Tyr163Cys)","volume":"176","author":"KE Hirabayashi","year":"2018","journal-title":"Am J Med Genet A"},{"issue":"5","key":"pone.0358119.ref009","doi-asserted-by":"crossref","first-page":"828","DOI":"10.1093\/hmg\/ddy394","article-title":"SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea\/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase","volume":"28","author":"L Holt-Danborg","year":"2019","journal-title":"Hum Mol Genet"},{"issue":"1","key":"pone.0358119.ref010","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1097\/MCD.0b013e328331de38","article-title":"Case of syndromic tufting enteropathy harbors SPINT2 mutation seen in congenital sodium diarrhea","volume":"19","author":"M Sivagnanam","year":"2010","journal-title":"Clin Dysmorphol"},{"issue":"2","key":"pone.0358119.ref011","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1007\/s12519-015-0070-y","article-title":"Genetic analysis of Italian patients with congenital tufting enteropathy","volume":"12","author":"M d\u2019Apolito","year":"2016","journal-title":"World J Pediatr"},{"issue":"3","key":"pone.0358119.ref012","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1097\/MCD.0b013e328361d42f","article-title":"Syndromic congenital diarrhea because of the SPINT2 mutation showing enterocyte tufting and unique electron microscopy findings","volume":"22","author":"MA Slae","year":"2013","journal-title":"Clin Dysmorphol"},{"key":"pone.0358119.ref013","doi-asserted-by":"crossref","DOI":"10.1136\/bcr-2016-217464","article-title":"Syndromic congenital diarrhoea: new SPINT2 mutation identified in the UAE","volume":"2017","author":"S Bou Chaaya","year":"2017","journal-title":"BMJ Case Rep"},{"issue":"12","key":"pone.0358119.ref014","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1007\/s12519-022-00613-6","article-title":"A novel SPINT2 missense mutation causes syndromic congenital sodium diarrhea","volume":"18","author":"X-X Zhang","year":"2022","journal-title":"World J Pediatr"},{"issue":"3","key":"pone.0358119.ref015","doi-asserted-by":"crossref","DOI":"10.1002\/ajmg.a.63474","article-title":"Biallelic variants of the first Kunitz domain of SPINT2 cause a non-syndromic form of congenital diarrhea and tufting enteropathy","volume":"194","author":"Y Al Rawahi","year":"2024","journal-title":"Am J Med Genet A"},{"issue":"7","key":"pone.0358119.ref016","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1007\/s00239-020-09959-9","article-title":"Evolutionary Aspects of the Structural Convergence and Functional Diversification of Kunitz-Domain Inhibitors","volume":"88","author":"M Mishra","year":"2020","journal-title":"J Mol Evol"},{"key":"pone.0358119.ref017","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1016\/j.micinf.2018.01.003","article-title":"Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival","volume":"20","author":"MTQ de Magalh\u00e3es","year":"2018","journal-title":"Microbes Infect"},{"issue":"7873","key":"pone.0358119.ref018","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"J Jumper","year":"2021","journal-title":"Nature"},{"key":"pone.0358119.ref019","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkad1011","article-title":"AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences","volume":"52","author":"M Varadi","year":"2024","journal-title":"Nucleic Acids Res"},{"issue":"9","key":"pone.0358119.ref020","doi-asserted-by":"crossref","first-page":"1779","DOI":"10.1042\/BCJ20200182","article-title":"Inhibition of an active zymogen protease: the zymogen form of matriptase is regulated by HAI-1 and HAI-2","volume":"477","author":"S Skovbjerg","year":"2020","journal-title":"Biochem J"},{"issue":"3","key":"pone.0358119.ref021","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.dci.2012.10.005","article-title":"Structure and function of invertebrate Kunitz serine protease inhibitors","volume":"39","author":"S Ranasinghe","year":"2013","journal-title":"Dev Comp Immunol"},{"issue":"18","key":"pone.0358119.ref022","doi-asserted-by":"crossref","first-page":"12202","DOI":"10.1074\/jbc.272.18.12202","article-title":"Identification and cloning of human placental bikunin, a novel serine protease inhibitor containing two Kunitz domains","volume":"272","author":"CW Marlor","year":"1997","journal-title":"J Biol Chem"},{"issue":"44","key":"pone.0358119.ref023","doi-asserted-by":"crossref","first-page":"27558","DOI":"10.1074\/jbc.272.44.27558","article-title":"Purification and cloning of hepatocyte growth factor activator inhibitor type 2, a Kunitz-type serine protease inhibitor","volume":"272","author":"T Kawaguchi","year":"1997","journal-title":"J Biol Chem"},{"issue":"43","key":"pone.0358119.ref024","doi-asserted-by":"crossref","first-page":"29495","DOI":"10.1074\/jbc.M801970200","article-title":"Potent inhibition and global co-localization implicate the transmembrane Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-2 in the regulation of epithelial matriptase activity","volume":"283","author":"R Szabo","year":"2008","journal-title":"J Biol Chem"},{"issue":"22","key":"pone.0358119.ref025","doi-asserted-by":"crossref","first-page":"4349","DOI":"10.1042\/BCJ20200630","article-title":"Insights into the regulation of the matriptase-prostasin proteolytic system","volume":"477","author":"L Holt-Danborg","year":"2020","journal-title":"Biochem J"},{"issue":"18","key":"pone.0358119.ref026","doi-asserted-by":"crossref","first-page":"12209","DOI":"10.1074\/jbc.272.18.12209","article-title":"Characterization of placental bikunin, a novel human serine protease inhibitor","volume":"272","author":"KA Delaria","year":"1997","journal-title":"J Biol Chem"},{"issue":"10","key":"pone.0358119.ref027","doi-asserted-by":"crossref","first-page":"105238","DOI":"10.1016\/j.jbc.2023.105238","article-title":"Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism","volume":"299","author":"CA Enns","year":"2023","journal-title":"J Biol Chem"},{"issue":"4","key":"pone.0358119.ref028","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0094267","article-title":"Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea","volume":"9","author":"N Faller","year":"2014","journal-title":"PLoS One"},{"key":"pone.0358119.ref029","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1515\/hsz-2024-0135","article-title":"GPI-anchored serine proteases: essential roles in development, homeostasis, and disease","volume":"406","author":"JG Lundgren","year":"2025","journal-title":"Biol Chem"},{"issue":"12","key":"pone.0358119.ref030","doi-asserted-by":"crossref","first-page":"3416","DOI":"10.1111\/febs.16399","article-title":"Understanding HAIs: Ally proteins in the fight against cancer","volume":"289","author":"AW Nonboe","year":"2022","journal-title":"FEBS J"},{"issue":"12","key":"pone.0358119.ref031","doi-asserted-by":"crossref","first-page":"3422","DOI":"10.1111\/febs.16348","article-title":"Role of the polycystic kidney disease domain in matriptase chaperone activity and localization of hepatocyte growth factor activator inhibitor-1","volume":"289","author":"F Yamashita","year":"2022","journal-title":"FEBS J"},{"issue":"36","key":"pone.0358119.ref032","doi-asserted-by":"crossref","first-page":"14867","DOI":"10.1074\/jbc.M117.775999","article-title":"Phosphorylation of the type II transmembrane serine protease, TMPRSS13, in hepatocyte growth factor activator inhibitor-1 and -2-mediated cell-surface localization","volume":"292","author":"AS Murray","year":"2017","journal-title":"J Biol Chem"},{"key":"pone.0358119.ref033","doi-asserted-by":"crossref","first-page":"123299","DOI":"10.1016\/j.lfs.2024.123299","article-title":"Intracellular autoactivation and surface location of hepsin, TMPRSS2, and TMPRSS13","volume":"361","author":"Z Jin","year":"2025","journal-title":"Life Sci"},{"issue":"9","key":"pone.0358119.ref034","doi-asserted-by":"crossref","first-page":"752","DOI":"10.1093\/hmg\/ddae005","article-title":"SPINT2 mutations in the Kunitz domain 2 found in SCSD patients inactivate HAI-2 as prostasin inhibitor via abnormal protein folding and N-glycosylation","volume":"33","author":"N Huang","year":"2024","journal-title":"Hum Mol Genet"},{"issue":"6","key":"pone.0358119.ref035","doi-asserted-by":"crossref","first-page":"918","DOI":"10.1016\/j.yexcr.2013.01.008","article-title":"Hepatocyte growth factor activator inhibitor-2 prevents shedding of matriptase","volume":"319","author":"BR Larsen","year":"2013","journal-title":"Exp Cell Res"},{"issue":"1","key":"pone.0358119.ref036","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1007\/s13577-021-00632-x","article-title":"The difference in the intracellular Arg\/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2","volume":"35","author":"N Huang","year":"2022","journal-title":"Hum Cell"},{"issue":"1","key":"pone.0358119.ref037","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1074\/jbc.RA118.004126","article-title":"Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors","volume":"294","author":"T Tamberg","year":"2019","journal-title":"J Biol Chem"},{"issue":"6","key":"pone.0358119.ref038","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1111\/tra.12482","article-title":"HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase","volume":"18","author":"AW Nonboe","year":"2017","journal-title":"Traffic"},{"issue":"7","key":"pone.0358119.ref039","doi-asserted-by":"crossref","first-page":"5793","DOI":"10.1074\/jbc.M110.186874","article-title":"Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase","volume":"286","author":"S Friis","year":"2011","journal-title":"J Biol Chem"},{"issue":"2","key":"pone.0358119.ref040","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1042\/BJ20071496","article-title":"Hepatocyte growth factor activator inhibitor-1 has a complex subcellular itinerary","volume":"413","author":"S Godiksen","year":"2008","journal-title":"Biochem J"},{"issue":"6","key":"pone.0358119.ref041","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.1074\/jbc.C115.706721","article-title":"Distinct Developmental Functions of Prostasin (CAP1\/PRSS8) Zymogen and Activated Prostasin","volume":"291","author":"S Friis","year":"2016","journal-title":"J Biol Chem"},{"issue":"1","key":"pone.0358119.ref042","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1186\/s12915-017-0384-4","article-title":"Matriptase zymogen supports epithelial development, homeostasis and regeneration","volume":"15","author":"S Friis","year":"2017","journal-title":"BMC Biol"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0358119","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T17:37:03Z","timestamp":1789493823000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0358119"}},"subtitle":[],"editor":[{"given":"Patrick","family":"Goymer","sequence":"first","affiliation":[{"name":"Public Library of Science","place":["UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND"]}],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,9,15]]},"references-count":42,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9,15]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0358119","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,15]]}}}