Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells.
The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.
D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001769:Blood; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D010053:Ovary / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism*; D014162:Transfection; D014841:von Willebrand Factor / Q000378:metabolism
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D001216:Asparagine; D005169:Factor VIII; D006026:Glycoside Hydrolases