B-domain deleted factor VIII is aggregated and degraded through proteasomal and lysosomal pathways

Abstract

Factor VIII (FVIII) processing within mammalian cells is demonstrated to be much less efficient than proteins of similar size. The deletion of the B-domain from FVIII improves the level of production, due partly to the increase in mRNA synthesis. We aimed to characterise the cellular fate and the intracellular processing of the FVIII molecule devoid of B-domain. A B-domain deleted factor VIII (BDD-FVIII) possessing a furin consensus cleavage site in the connecting segment between the heavy and the light chain, was produced in CHO cell line. In such cells, FVIII was retained as two single chain products from which a majority was aggregated. The two species were located in Triton X-100 soluble (for 60-80%) and insoluble fractions (for 20-40%). The incubation of the expressing cells with tunicamycin (5 mug/ml) and the treatment of the intracellular species with a mixture of Neuraminidase and N-glycosidase-F revealed that both intracellular species were N-glycosylated. Furin over-expression neither diminished the intracellular FVIII contents nor improved its extracellular production. Intracellular FVIII was degraded through both lysosomal and proteasomal pathways as evidenced by inhibitor treatments (e.g. NH(4)Cl, leupeptin, clasto-Lactacystin beta-lactone and MG-132), pulse-chase analysis and confocal observations. This study demonstrates that a BDD-FVIII expressed in CHO cells is inefficiently processed consecutively to intracellular aggregation, proteasomal degradation, and routage to lysosomes.

Cellular Processing of Myocilin

“…Misfolded or inappropriately expressed proteins accumulated in the ER are also likely to be degraded in the lysosome compartment [51]. In the literature, proteins including gap junction proteins connexins [42], [52], epidermal growth factor [53], and factor VIII [54] have been shown to utilize both proteasomal and lysosomal systems for their processing and degradation. Myocilin can now be added to the list.…”

Protein-Engineered Coagulation Factors for Hemophilia Gene Therapy

“…Although FVIII-BDD was designed to maintain furin processing under the untested assumption that heterodimer formation was important for biological activity, attempts to enhance furin cleavage in fact modestly decreased FVIII-BDD secretion 116 . In a comprehensive study of the role of furin in FVIII biology, FVIII-BDD bioengineered to avoid furin processing by deleting the cleavage sequence (FVIII-ΔF; Figure 1B) actually enhanced FVIII secretion 3-fold from both mammalian cell culture and after AAV-based gene therapy in small- and large-animal HA models 117 .…”

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

“…Swaroop et al have shown that FVIII interacts with the chaperones immunoglobulin-binding protein (BiP/GRP78) [15], calnexin, and calreticulin [16], which results in formation of non-disulfide-bonded high molecular weight aggregates in the ER. These misfolded FVIII proteins are subsequently designated for proteasomal and lysosomal degradation [17]. Nevertheless, a number of different modifications in recombinant FVIII have helped increase secretion efficiency.…”