The Carboxyl-Terminal Region of Factor IX Is Essential for Its Secretion † Sumiko Kurachi, Dennis P. Pantazatos, and Kotoku Kurachi* Department of Human Genetics, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109-0618 ReceiVed August 12, 1996; ReVised Manuscript ReceiVed January 21, 1997 X ABSTRACT: 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 µM 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 µM), 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 (e10%). 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. Factor IX is a plasma glycoprotein which plays an essential role in the middle phase of the blood coagulation cascade (Kurachi et al., 1993; Saito, 1991). Its deficiency or reduced functional level results in an abnormal bleeding disorder, hemophilia B, with various levels of severity (Kurachi et al., 1993; Giannelli et al., 1994). Since its normal gene was isolated and completely sequenced a decade ago (Yoshitake et al., 1985), almost 1400 abnormal human factor IX genes have been studied at the molecular level, producing a wealth of knowledge on the structure-function relationship of the factor IX gene and protein (Giannelli et al., 1994, 1996). This makes factor IX one of the most thoroughly studied mammalian proteins. The factor IX database (Giannelli et al., 1994, 1996) shows that mutations in two regions, one spanning aa 1 sequence 191-198 and the other spanning the carboxyl-terminal (C- terminal) region aa 403-415 and a significant number of randomly scattered mutations present a unique class of hemophilia B. Most of these mutations display severely reduced factor IX antigen and activity levels in the circula- tion. The sequence region aa 191-198 is a part of the hydrophobic core of the catalytic subunit. Understandably, most, but not all of the randomly distributed mutations are nonsense mutations or mutations at splicing sites (Giannelli et al., 1994). The resulting phenotype is analogous to that † This work was supported in part by grants from the National Institutes of Health, HL38644, the University of Michigan Multipurpose Arthritis Center Grant NIH 5P60AR20557, and the University of Michigan General Clinical Research Center Grant M01RR0042. * To whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, April 1, 1997. 1 Abbreviations: aa, amino acid; C-terminal, carboxy terminal; PCR, polymerase chain reaction; DMEM, Dulbecco’s modified eagle me- dium; FCS, fetal calf serum; ALLM, N-acetyl-L-leucinyl-L-leucinyl- L-methioninal; ALLN, N-acetyl-L-leucinyl-L-lencinyl-L-norleucinal; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; TLCK, N-tosyl- L-lysyl chloromethyl ketone; CNBr, cyanogen bromide; ELISA, enzyme-linked immunosorbent assay; ds, double stranded; ss, single stranded; nt, nucleotide; bp, base pairs; PBS, phosphate-buffered saline; ER, endoplastic reticulum. 4337 Biochemistry 1997, 36, 4337-4344 S0006-2960(96)02002-8 CCC: $14.00 © 1997 American Chemical Society