Abstract
Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Analysis of Variance
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Animals
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Betaine / metabolism
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Betaine / pharmacology
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Blotting, Western
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CHO Cells
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Cricetinae
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Cricetulus
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Factor VIII / genetics
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Factor VIII / metabolism*
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Flow Cytometry
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Genetic Vectors
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Hemophilia A / drug therapy
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Hemophilia A / metabolism*
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Humans
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Lentivirus
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Microscopy, Fluorescence
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Molecular Chaperones / metabolism*
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Protein Folding
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Protein Transport / drug effects
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Protein Transport / physiology
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Recombinant Proteins / biosynthesis
Substances
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Molecular Chaperones
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Recombinant Proteins
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Betaine
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Factor VIII
Grants and funding
SR, DA, CU and PQL are students within the graduate study program GK-1172 funded by the Deutsche Forschungsgemeinschaft. The research was supported by the Stiftung Hämotherapie-Forschung e.V. to JS. ES and TT received funding through the Excellence Cluster Cardio-Pulmonary System (ECCPS, Deutsche Forschungsgemeinschaft). ES and JS received support from the Landesoffensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE)-Center for Cell and Gene Therapy, Frankfurt, funded by the Hessisches Ministerium für Wissenschaft und Kunst, funding reference number: III L 4–518/17.004 (2010). JS receives funding from Bayer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.