The carboxyl-terminal region of factor IX is essential for its secretion.

The carboxyl-terminal region of factor IX (residues 403-415) contains several natural mutations which result in mild to severe forms of hemophilia B. A battery of factor IX minigene expression vectors carrying various mutations in the C-terminal region were constructed and studied by transient expression assay using HepG2 cells. Mutations included in this study are Y404P, I408N, T412N, T412S, T415G, T415S, T415L, and T415R as well as five selected naturally occurring mutations in the region, R403Q, R403W, Y404H, W407R, and T412K. In comparison to the normal factor IX, these mutations neither significantly affected the factor IX mRNA level nor affected the stability of the secreted factor IX in the culture medium but did decrease to various extents the intracellular and secreted factor IX protein levels as quantified by enzyme-linked immunosorbent assay. T415L, T415S, and T415R showed only minor reductions in the intracellular and minor to moderate reductions in the secreted factor IX levels. T415G showed only minor reduction in the intracellular factor IX level but substantial reduction in the secreted levels. R403Q, R403W, and T412S showed moderate reductions in both intracellular and secreted factor IX levels. Y404H, Y404P, W407R, I408N, T412K, and T412N also showed minor to moderate reductions in the intracellular factor IX levels but very severe reductions in the secreted factor IX level. In one stage clotting assays, secreted factor IX mutants showed specific activities very similar to that of the normal factor IX, suggesting that the carboxyl-terminal region is neither directly involved in the tenase complex formation required for factor X activation nor involved in the activation of factor IX itself. In comparison to the normal factor IX, secreted levels of factor IX mutants with mutations R403Q, Y404H, W407R, and T412K were also very similar to the plasma levels reported for the hemophilia B patients carrying the same mutations. Treatment of cells with proteasome inhibitors (ALLM and ALLN) added to the culture medium at 50 microM resulted in drastic increases of the intracellular mutant factor IX to the levels equivalent to that of the normal factor IX, while the secreted factor IX levels were little or only marginally affected. With a higher concentration of the inhibitors (100 microM), however, both the intracellular and secreted mutant factor IX were fully elevated to the normal factor IX levels. Intracellular and secreted levels of the normal factor IX were little affected by the low inhibitor concentration and only marginally, if at all, at the higher concentration (< or = 10%). Serine protease inhibitors did not significantly affect intracellular and secreted factor IX levels. These results indicate that the carboxyl-terminal region plays a critical role in the cellular secretion of factor IX and that the mutant factor IX proteins carrying specific mutations in this region are subjected to efficient elimination by the proteasome protein degradation mechanism. Furthermore, these results strongly support that HepG2 cells can be utilized as a robust in vitro assay system for studying factor IX biosynthesis, well mimicking the in vivo phenomenon.