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.
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March 2018
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The human immunodeficiency virus. In this issue of the Biochemical Journal, Parajuli et al. report that restricted the HIV Env (envelope glycoprotein) glycan engagement by a lectin reengineered DAVEI protein chimera is sufficient for virolysis. For details, see pages 931–957.
Research Article|
March 06 2018
Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII
Wei Wei;
Wei Wei
1Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A.
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Saurav Misra;
Saurav Misra
2Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS, U.S.A.
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Matthew V. Cannon;
Matthew V. Cannon
*
1Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A.
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Renchi Yang;
Renchi Yang
3State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China
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Xiaofan Zhu;
Xiaofan Zhu
3State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China
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Reid Gilmore;
Reid Gilmore
4Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, U.S.A.
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Min Zhu;
Min Zhu
5Department of Pathology, Karamay Central Hospital, Karamay, Xinjiang, China
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Bin Zhang
Bin Zhang
1Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH, U.S.A.
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Publisher: Portland Press Ltd
Received:
November 21 2017
Revision Received:
February 07 2018
Accepted:
February 14 2018
Accepted Manuscript online:
February 14 2018
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2018
Biochem J (2018) 475 (5): 873–886.
Article history
Received:
November 21 2017
Revision Received:
February 07 2018
Accepted:
February 14 2018
Accepted Manuscript online:
February 14 2018
Citation
Wei Wei, Saurav Misra, Matthew V. Cannon, Renchi Yang, Xiaofan Zhu, Reid Gilmore, Min Zhu, Bin Zhang; Molecular mechanisms of missense mutations that generate ectopic N-glycosylation sites in coagulation factor VIII. Biochem J 15 March 2018; 475 (5): 873–886. doi: https://doi.org/10.1042/BCJ20170884
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