Differential Engagement of the Unfolded Protein Response by Human and Porcine Factor VIII

Abstract 2224

Despite 83% amino acid sequence identity, human factor VIII (fVIII) and porcine fVIII display a striking species-specific expression differential (10 – 100- fold) that may be relevant to each species’ maintenance of hemostasis. This expression differential has been exploited for the development of improved recombinant fVIII therapeutics and gene-transfer-based therapies (Doeringet al., JBC 2002, JBC 2004; Gangadharanet al., Blood 2006; Doeringet al., Mol. Ther. 2007; Doeringet al., Mol. Ther. 2009; Doorisset al., Hum. Gene Ther. 2009). The exact biochemical or cellular mechanisms responsible for this phenomenon has yet to be uncovered. However, the expression differential has been temporally and spatially localized to post-translational steps that occur in the endoplasmic reticulum (ER)/golgi apparatus (Doeringet al., JBC 2004). The unfolded protein response (UPR) is a coordinated cellular mechanism designed to regulate the build-up and flow of proteins in the ER and is sometimes referred to as the “quality control pathway”. Early studies of recombinant fVIII production in heterologous cell culture systems uncovered that human fVIII biosynthesis induces up-regulation of the UPR through interaction with immunoglobulin binding protein (BiP)/glucose regulated protein 78 (GRP78). These studies prompted the speculation that human fVIII may not undergo correct secondary and tertiary folding efficiently or, alternatively, may display thermodynamic instability subsequent to initial folding. Either event could serve to induce UPR activity and affect overall fVIII biosynthesis. In the current study, we hypothesized that the differential in biosynthesis between recombinant human and porcine fVIII results from differential engagement of the UPR pathway. To test this hypothesis, we studied the UPR response during expression of recombinant human and porcine fVIII in heterologous baby hamster kidney cells. In experiments utilizing a ER response element (ERSE)-luciferase reporter construct, human fVIII expression was shown to increase luciferase activity 2-fold over that observed with porcine fVIII despite the secretion of 10-fold more porcine fVIII based on activity measurement of the conditioned medium. Human fVIII expression also induced X-box protein 1 (XBP-1) splicing, another marker of UPR activation, to a greater extent than did porcine fVIII expression (2-fold versus 1.1-fold over naïve BHK cells, respectively). Immunofluorescence microscopy of fVIII expressing cells revealed that human fVIII almost exclusively was localized to the ER and displayed significant co-localization with BiP, a resident ER luminal protein. In contrast, a significant proportion of porcine fVIII antigen was localized to the Golgi apparatus and co-localized with the lectin, wheat germ agglutinin (r² = 0.8 versus 0.6 for human fVIII), suggesting more efficient transit through the secretory pathway. Perturbation of the intracellular BiP levels by transduction of fVIII expressing BHK cells with recombinant lentivector encoding BiP revealed that, under normal conditions, BiP levels may actually be limiting in terms of the interaction with human fVIII. Over-expression of BiP (6.5-fold increased BiP mRNA levels) reduced the secretion of human fVIII by 50%, whereas the same BiP over-expression had no affect on porcine fVIII biosynthesis. In neither case were fVIII mRNA levels negatively affected by BiP over-expression. In contrast, transduction of fVIII expressing BHK cells with lentivector encoding shRNA targeting BiP led to 2-fold improvement in human fVIII expression levels. Again, these data support a role of BiP in the impediment of human fVIII biosynthesis that is not equivalent for porcine fVIII. Therefore, differential engagement of the UPR by human and porcine fVIII was observed in the current study, thus supporting a dominant role of protein folding/stability characteristics in the expression differential present among porcine and human fVIII. This study also demonstrates that the investigation of orthologous protein biosynthesis can yield important insights into the regulation of gene product levels through non-traditional mechanisms.