{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,27]],"date-time":"2025-10-27T05:03:51Z","timestamp":1761541431910},"reference-count":51,"publisher":"Portland Press Ltd.","issue":"5","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2018,3,15]]},"abstract":"<jats:p>N-glycosylation is a common posttranslational modification of secreted and membrane proteins, catalyzed by the two enzymatic isoforms of the oligosaccharyltransferase, STT3A and STT3B. Missense mutations are the most common mutations in inherited diseases; however, missense mutations that generate extra, non-native N-glycosylation sites have not been well characterized. Coagulation factor VIII (FVIII) contains five consensus N-glycosylation sites outside its functionally dispensable B domain. We developed a computer program that identified hemophilia A mutations in FVIII that can potentially create ectopic glycosylation sites. We determined that 18 of these ectopic sites indeed become N-glycosylated. These sites span the domains of FVIII and are primarily associated with a severe disease phenotype. Using STT3A and STT3B knockout cells, we determined that ectopic glycosylation exhibited different degrees of dependence on STT3A and STT3B. By separating the effects of ectopic N-glycosylation from those due to underlying amino acid changes, we showed that ectopic glycans promote the secretion of some mutants, but impair the secretion of others. However, ectopic glycans that enhanced secretion could not functionally replace a native N-glycan in the same domain. Secretion-deficient mutants, but not mutants with elevated secretion levels, show increased association with the endoplasmic reticulum chaperones BiP (immunoglobulin heavy chain-binding protein) and calreticulin. Though secreted to different extents, all studied mutants exhibited lower relative activity than wild-type FVIII. Our results reveal differential impacts of ectopic N-glycosylation on FVIII folding, trafficking and activity, which highlight complex disease-causing mechanisms of FVIII missense mutations. Our findings are relevant to other secreted and membrane proteins with mutations that generate ectopic N-glycans.<\/jats:p>","DOI":"10.1042\/bcj20170884","type":"journal-article","created":{"date-parts":[[2018,2,15]],"date-time":"2018-02-15T01:25:24Z","timestamp":1518657924000},"page":"873-886","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII"],"prefix":"10.1042","volume":"475","author":[{"given":"Wei","family":"Wei","sequence":"first","affiliation":[{"name":"Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saurav","family":"Misra","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, U.S.A."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew V.","family":"Cannon","sequence":"additional","affiliation":[{"name":"Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Renchi","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaofan","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reid","family":"Gilmore","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, U.S.A."}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Min","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Pathology, Karamay Central Hospital, Karamay, Xinjiang, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bin","family":"Zhang","sequence":"additional","affiliation":[{"name":"Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A."}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2018,3,6]]},"reference":[{"key":"2021112217001939800_BCJ-2017-0884C1","doi-asserted-by":"publisher","first-page":"2364","DOI":"10.1126\/science.291.5512.2364","article-title":"Intracellular functions of N-linked glycans","volume":"291","author":"Helenius","year":"2001","journal-title":"Science"},{"key":"2021112217001939800_BCJ-2017-0884C2","doi-asserted-by":"publisher","first-page":"272","DOI":"10.1016\/j.cell.2008.11.047","article-title":"Cotranslational and posttranslational N-glycosylation of polypeptides by distinct mammalian OST isoforms","volume":"136","author":"Ruiz-Canada","year":"2009","journal-title":"Cell"},{"key":"2021112217001939800_BCJ-2017-0884C3","doi-asserted-by":"publisher","first-page":"3379","DOI":"10.1016\/j.febslet.2015.07.021","article-title":"A sweet code for glycoprotein folding","volume":"589","author":"Caramelo","year":"2015","journal-title":"FEBS Lett."},{"key":"2021112217001939800_BCJ-2017-0884C4","doi-asserted-by":"publisher","first-page":"57","DOI":"10.1016\/j.ceb.2016.03.021","article-title":"N-linked glycosylation and homeostasis of the endoplasmic reticulum","volume":"41","author":"Cherepanova","year":"2016","journal-title":"Curr. 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