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

Jean-Luc Plantier; Benoit Guillet; Cécile Ducasse; Nathalie Enjolras; Marie-Hélène Rodriguez; Véronique Rolli; Claude Négrier

doi:10.1160/TH04-09-0579

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 93(05): 824-832
DOI: 10.1160/TH04-09-0579

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

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

Authors

Jean-Luc Plantier

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France
Benoit Guillet

²Centre de traitement des maladies hémorragiques, Hôpital Bicêtre, Le Kremlin-Bicêtre, France
Cécile Ducasse

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France
Nathalie Enjolras

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France
Marie-Hélène Rodriguez

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France
Véronique Rolli

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France
Claude Négrier

¹Laboratoire d’ Hémobiologie-Faculté de Médecine RTH Laennec, Lyon, France

Abstract

Summary

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 μg/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. NH₄Cl, leupeptin, clasto-Lactacystin β-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.

Keywords

Degradation - factor VIII - furin - lysosome

Full Text

References

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