Confocal microscopy analysis of native, full length and B-domain deleted coagulation factor VIII trafficking in mammalian cells
Sven Becke (1), Jeremy C. Simpson (2), Rainer Pepperkok (2), Stefan Heinz (1), Christian Herder (1, 3), Manuel Grez (3), Erhard Seifried (1),Torsten Tonn (1)
(1) Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Frankfurt/Main, Germany (2) European Molecular Biology Laboratory (EMBL), Heidelberg, Germany (3) Chemotherapeutisches Forschungsinstit
Summary In mammalian cells, factor VIII (FVIII) secretion depends uponits interaction with chaperones of the endoplasmic reticulum(ER) and requires a unique ATP-dependent step to dissociateaggregates formed within the ER. To further elucidate mechanismswhich might account for the inefficient secretion ofrecombinant FVIII (rFVIII), we have analyzed the pathways ofrecombinant full length (rFVIII-FL) and B-domain deleted(rFVIII?B) FVIII and compared these to the secretion route ofnative FVIII in primary hepatocytes. Using confocal laser scanningmicroscopy in combination with a pulse chase of a knownsecretion marker, we describe the trafficking route of FVIII,which upon release from the ER - where it colocalizes withcalnexin - is transported to the Golgi complex in vesiculartubulartransport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion ofrFVIII is retained in the ER and additionally in structures whichcould not be assigned to the ER, Golgi complex or intermediatecompartment. Moderate BiP transcription levels indicatethat this observed retention of FVIII does not reflect cellularstress due to an overexpression of FVIII-protein in transducedcells. Moreover, a pulse of newly synthesized rFVIII protein isreleased within 4 hrs, indicating that once rFVIII is releasedfrom the ER there is no further limitation to its secretion. Ourdata provide new details about the secretory route of FVIII,which may ultimately help to identify factors currently limitingthe efficient and physiological expression of FVIII in genetherapy and manufacture.