Abstract
Blood coagulation factor VIII (FVIII) is a key cofactor in regulation of blood coagulation. This study investigated the mechanism by which FVIII is translated and transported into the endoplasmic reticulum (ER) and processed in the Golgi apparatus before secretion using an in vitro cell model. HEK-293T cells were transfected with vectors carrying wild-type (WT) FVIII or polymorphic FVIII D1241E for coexpression with ER lectins and treatment with tunicamycin (an N-linked glycosylation inhibitor), 1-deoxynojirimycin (an alpha-glucosidase inhibitor), endoglycosidase H, or MG132 (Cbz-Leu-Leu-leucinal; a proteasome inhibitor). The data showed that the minor allele of FVIII D1241E was able to reduce FVIII secretion into the conditioned medium but maintain a normal level of procoagulation ability, although both FVIII WT and the minor allele of FVIII D1241E showed similar levels of transcription and translation capacities. Functionally, the D1241E polymorphism led to a reduced level of FVIII in the Golgi apparatus because of its reduced association with malectin, which interacts with newly synthesized glycoproteins in the ER for FVIII folding and trafficking, leading to degradation of the minor allele of FVIII D1241E in the cytosol. This study demonstrated that malectin is important for regulation of the FVIII posttranslational process and that the minor allele of FVIII D1241E had a reduced association with malectin but an increased capacity for proteasomal FVIII degradation. These data imply the role of the ER quality control in future recombinant FVIII development.