Becker Sven, Simpson Jeremy C, Pepperkok Rainer, Heinz Stefan, Herder Christian, Grez Manuel, Seifried Erhard, Tonn Torsten
Institute for Transfusion Medicine and Immunohematology, Red Cross Blood Donor Service Baden-Wuerttemberg-Hesse, Sandhofstr. 1, D-60528 Frankfurt, Germany.
Thromb Haemost. 2004 Jul;92(1):23-35. doi: 10.1160/TH03-06-0360.
In mammalian cells, factor VIII (FVIII) secretion depends upon its interaction with chaperones of the endoplasmic reticulum (ER) and requires a unique ATP-dependent step to dissociate aggregates formed within the ER. To further elucidate mechanisms which might account for the inefficient secretion of recombinant FVIII (rFVIII), we have analyzed the pathways of recombinant full length (rFVIII-FL) and B-domain deleted (rFVIII Delta B) FVIII and compared these to the secretion route of native FVIII in primary hepatocytes. Using confocal laser scanning microscopy in combination with a pulse chase of a known secretion marker, we describe the trafficking route of FVIII, which upon release from the ER--where it colocalizes with calnexin--is transported to the Golgi complex in vesicular-tubular transport complexes (VTCs) which could be further identified as being COP I coated. However, a large portion of rFVIII is retained in the ER and additionally in structures which could not be assigned to the ER, Golgi complex or intermediate compartment. Moderate BiP transcription levels indicate that this observed retention of FVIII does not reflect cellular stress due to an overexpression of FVIII-protein in transduced cells. Moreover, a pulse of newly synthesized rFVIII protein is released within 4 hrs, indicating that once rFVIII is released from the ER there is no further limitation to its secretion. Our data provide new details about the secretory route of FVIII, which may ultimately help to identify factors currently limiting the efficient and physiological expression of FVIII in gene therapy and manufacture.
在哺乳动物细胞中，凝血因子VIII（FVIII）的分泌依赖于它与内质网（ER）伴侣蛋白的相互作用，并且需要一个独特的ATP依赖步骤来解离在内质网中形成的聚集体。为了进一步阐明可能解释重组FVIII（rFVIII）分泌效率低下的机制，我们分析了重组全长（rFVIII-FL）和B结构域缺失（rFVIII ΔB）FVIII的途径，并将这些与原代肝细胞中天然FVIII的分泌途径进行了比较。使用共聚焦激光扫描显微镜结合已知分泌标记物的脉冲追踪，我们描述了FVIII的运输途径，FVIII从与钙连接蛋白共定位的内质网释放后，通过囊泡管状运输复合体（VTCs）被运输到高尔基体复合体，这些复合体可进一步鉴定为被COP I包被。然而，大部分rFVIII保留在内质网中，此外还保留在无法归属于内质网、高尔基体复合体或中间区室的结构中。适度的BiP转录水平表明，观察到的FVIII保留并不反映由于转导细胞中FVIII蛋白过表达导致的细胞应激。此外，新合成的rFVIII蛋白在4小时内释放，这表明一旦rFVIII从内质网释放，其分泌就不再有进一步的限制。我们的数据提供了关于FVIII分泌途径的新细节，这最终可能有助于识别目前在基因治疗和生产中限制FVIII高效和生理性表达的因素。
