A 110-AMINO ACID REGION WITHIN THE A1-DOMAIN OF COAGULATION-FACTOR-VIII INHIBITS SECRETION FROM MAMMALIAN-CELLS

Factor VIII is the coagulation factor deficient in the X-chromosome li nked bleeding disorder hemophilia A. Factor VIII is homologous to bloo d coagulation factor V, both having a domain structure of A1-A2-B-A3-C 1-C2. Previous transfection studies demonstrated that factor VIII is 1 0-fold less efficiently expressed than the homologous coagulation fact or, factor V, The inefficient expression correlated with interaction o f the factor VIII primary translation product with the protein chapero nin BiP in the lumen of the endoplasmic reticulum, In contrast, factor V was not detected in association with BiP and was secreted efficient ly, To determine whether specific amino acid sequences within factor V III inhibit secretion, we have studied the secretion of factor VIII de letion and factor VIII/factor V chimeric proteins upon transient trans fection of COS-1 monkey cells, A chimeric factor VIII protein that con tained the A1- and A2-domains of factor V was secreted with a similar efficiency as wild-type factor V, whereas the complementary chimera ha ving the A1- and A2-domains of factor VIII was secreted with low effic iency, similar to wild-type factor WI, These results suggested that se quences within the A1- and A2-domains were responsible for the low sec retion efficiency of factor VIII, Secretion of A1-domain deleted facto r VIII was increased approximately 10-fold compared to wild-type facto r VIII or A2-domain-deleted factor VIII, Expression of the factor VIII Al-domain alone did not yield secreted protein, whereas expression of the factor VIII A2-domain alone or the factor V A1-domain or A2-domai n alone directed synthesis of secreted protein,. Secretion of a hybrid in which the carboxyl-terminal 110 amino acids of the A1-domain were replaced by homologous sequences from the factor V A1-domain was also increased 10-fold compared to wild type factor VIII, however, the secr eted protein was not functional and the heavy and fight chains were no t associated. These results localize a 1l0-amino acid region within th e Al domain that inhibits factor VIII secretion, This region is cluste red with multiple short peptide se sequences that have potential to bi nd BiP,