ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion.
Deficiency in coagulation factor VIII leads to the bleeding disorder hemophilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this report, we identified that this ATP-dependent rate-limiting step involves the dissociation of non-disulfide-linked aggregates within the endoplasmic reticulum (ER). In contrast to the numerous examples of interchain disulfide-linked aggregates, factor VIII is the first protein characterized to form non-disulfide-linked high molecular weight aggregates within the ER. Approximately a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional factor VIII appeared in the medium. The aggregated complexes did not require proteasomal degradation for clearance. Aggregate formation was enhanced by ATP depletion, and upon restoration of metabolic energy, these aggregates were dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting that these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII that was aggregated. In addition, a 110 amino acid sequence containing a hydrophobic beta-sheet within factor VIII was identified that may predispose factor VIII to aggregation. These data show that formation and ATP-dependent dissolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate-limiting step during the folding process in the early secretory pathway. In summary, we have identified an unprecedented requirement for protein transport out of the ER that involves an ATP-dependent dissociation of non-disulfide-linked aggregates within the ER.
Tagliavacca
L
L
Department of Biological Chemistry and Howard Hughes Medical Institute, 1150 West Medical Center Drive, University of Michigan Medical Center, Ann Arbor, Michigan 48109, USA.
Wang
Q
Q
Kaufman
R J
RJ
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D004220:Disulfides / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D005169:Factor VIII / Q000378:metabolism*; D006360:Heat-Shock Proteins; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014841:von Willebrand Factor / Q000096:biosynthesis
D002352:Carrier Proteins; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D014841:von Willebrand Factor; C067713:lactacystin; D000255:Adenosine Triphosphate; D005169:Factor VIII; D000111:Acetylcysteine