Proc. Natl. Acad. Sci. USA Vol. 94, pp. 13654–13660, December 1997 Cell Biology This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 30, 1996. Structural features of the kringle domain determine the intracellular degradation of under-g-carboxylated prothrombin: Studies of chimeric ratyhuman prothrombin WEI WU*, JOHN D. BANCROFT†, AND J. W. SUTTIE*‡ *Department of Biochemistry, College of Agricultural and Life Sciences, and †Department of Pediatrics, The Medical School, University of Wisconsin–Madison, Madison, WI 53706 Contributed by J. W. Suttie, October 3, 1997 ABSTRACT Vitamin K antagonists such as warfarin in- of prothrombin secretion, and an intracellular accumulation of hibit the vitamin K-dependent g-glutamyl carboxylation dur- under-g-carboxylated prothrombin that is degraded in a pre- ing protein processing and block the secretion of under-g- Golgi compartment (5–7). In patients treated with oral anti- carboxylated prothrombin (FII) in the rat but not in the coagulants (4) and in cultures of a human hepatoma (HepG2) human or bovine. Under-g-carboxylated prothrombin is also cell line incubated with warfarin (8), under-g-carboxylated secreted from warfarin-treated human (HepG2) cell cultures prothrombin is secreted. Rat prothrombin (rFII) stably ex- but is degraded in the endoplasmic reticulum in warfarin- pressed in HepG2 cells treated with warfarin is not secreted treated rat (H-35) cell cultures. This differential response to but is degraded intracellularly (8), whereas secretion of en- warfarin has been shown to be determined by the structural dogenous human prothrombin (hFII) is not altered. These difference in the proteins rather than by the origin of the cell data suggest that a structural element within the prothrombin line. When recombinant rat prothrombin (rFII) and human molecule determines the fate (secretion vs. retention and prothrombin (hFII) were expressed in a transformed human degradation) of the under-g-carboxylated protein during its kidney cell line (HEK293), secretion of rFII but not hFII was intracellular processing through the secretory pathway. drastically decreased in response to warfarin. To determine Mature plasma prothrombin consists of an amino-terminal the structural signal required for this differential response, Gla domain, a kringle domain containing two kringle struc- chimeric cDNAs with the propeptideyGla domains, kringle tures, and a carboxyl-terminal serine protease catalytic domain domain, and serine protease domain exchanged between rFII (9). To define the structural signal present in prothrombin that y and hFII were generated (FIIRHH and FIIHRR, FIIRRH and is responsible for the differential processing of rat human y FIIHHR, FIIRHR and FIIHRH) and expressed in both warfarin- protein in response to warfarin, a number of chimeric rFII treated HEK293 cells and HepG2 cells. The presence of the hFII cDNAs were generated by using recombinant DNA hFII kringle domain changed the stability of rFII to that of techniques. These constructs were expressed in human em- hFII, and the rFII kringle domain changed the stability of hFII bryonic kidney (HEK293) and hepatoma (HepG2) cell lines. to that of rFII. The kringle domain therefore is critical in The response of these chimeras to warfarin has been evaluated determining the metabolic fate of under-g-carboxylated pro- to determine the location of the structural difference between thrombin precursors during processing. Prothrombin con- rat and human prothrombin that is responsible for their tains two kringle structures, and expression of additional differential processing. rFIIyhFII chimeras (FIIHrhH and FIIHhrH, FIIRrhR, and FIIRhrR) was used to determine that the first of the two MATERIALS AND METHODS kringles plays a more important role in the recognition process. Chimeric cDNA Constructs. The cDNAs coding for rFII and hFII were cloned into pcDNA3 as EcoRI fragments as previ- ously described (8). Chimeric cDNA construction was based During biosynthesis, specific glutamyl residues of prothrombin on the PCR. Oligonucleotides PM1 to PM15 were designed (coagulation factor II) are carboxylated to g-carboxyglutamyl with PcDNA3-rFII and hFII as templates, with necessary (Gla) residues by a vitamin K-dependent hepatic microsomal restriction sites at 59 ends. BbsI class II restriction sites were enzyme (1). The coproduct of this reaction, vitamin K 2,3- created to ensure no disruption of the native sequence at the epoxide is reduced to the enzymatically active hydronaphtho- ligation junction sites (10). All fragments generated by PCR quinone form of the vitamin by a microsomal epoxide reduc- were sequenced to ensure no mutations were introduced. The tase (1). Warfarin, a 4-hydroxycoumarin-based anticoagulant, constructs are shown in Fig. 4. blocks g-carboxylation by inhibiting the recycling of vitamin K PcDNA3-FII and PcDNA3-FII . A 0.27-kb fragment from its epoxide form to the reduced form (2). In the acquired HRR RHH containing the HFII propeptide and Gla sequences was am- vitamin K deficiency produced in the presence of warfarin, plified from pcDNA3-HFII with PM1 and PM3, subcloned under-g-carboxylated forms of prothrombin appear in the into pBluescript (1y2) to give pblysk-PG . Primers PM4 and plasma of some species (3, 4). H PM5 were used to amplify a 0.3-kb RFII fragment from Secretion of under-g-carboxylated prothrombin has been pcDNA3-RFII, and the BbsIyApaI digest of the PCR product shown to be species-dependent. In the rat or in a rat hepatoma was ligated with the PG insert (BamHIyBbsI) of pblysk-PG cell line (H-35), warfarin treatment results in a drastic decline H H ‡To whom reprint requests should be addressed at: University of © 1997 by The National Academy of Sciences 0027-8424y97y9413654-7$2.00y0 Wisconsin–Madison, Department of Biochemistry, 420 Henry Mall, PNAS is available online at http:yywww.pnas.org. Madison, WI 53706-1569. e-mail: [email protected]. 13654 Downloaded by guest on September 24, 2021 Cell Biology: Wu et al. Proc. Natl. Acad. Sci. USA 94 (1997) 13655 9 y and the 3 end ApaI EcoRI fragment (1.46 kb) of RFII into FIIRhrR) in HepG2 cells, the specific anti-rFII antibody was used. g pcDNA3 to give pcDNA3-FIIHRR. A 0.37-kb fragment con- Citrate-washed BaSO4 was used to adsorb fully -carboxylated taining the RFII propeptide and Gla sequences was amplified prothrombin, and under-g-carboxylated prothrombin was de- from pcDNA3-RFII with PM1 and PM2 and subcloned into fined as prothrombin remained in the supernatant after BaSO4 1y2 y pBluescript ( ) to generate pbl sk-PGR. PM4 and PM6 adsorption (8). Total protein secretion was measured by 10% were used to amplify a 0.47-kb HFII fragment from pcDNA3- trichloroacetic acid precipitation of cell media followed by quan- HFII, and the BbsIyBstEII digest of the PCR product was titation of precipitated radioactivity with liquid scintillation spec- y y ligated with the PGR insert (BamHI BbsI) of pbl sk-PGR and trometry. the 39 end BstEIIyEcoRI fragment (1.3 kb) of HFII into Drug Treatment of the Cells. In steady-state metabolic labeling pcDNA3 to give pcDNA3-FIIRHH. experiments, HepG2 and HEK293 cells were treated with vita- PcDNA3-FIIHHR, PcDNA3-FIIRRH, PcDNA3-FIIHRH, min K (10 mg phylloquinoneyml) or warfarin (1 mgyml) in the PcDNA3-FIIRHR, PcDNA3-FIIHrhH, PcDNA3-FIIHhrH, presence of [35S]methionine for 24 h. In pulse-labeling experi- PcDNA3-FIIRhrR, and PcDNA3-FIIRrhR. The following se- ments, cells first were treated with vitamin K (10 mgyml) or quences were amplified as the 0.7-kb human kringle (KH) with warfarin (1 mgyml) for 24 h before 5-min pulse labeling in the PM4 and PM10, the 0.7-kb rat kringle (KR) with PM4 and presence of vitamin K or warfarin. In some experiments, brefel- PM9, the 1.0-kb human protease domain (PH) with PM7 and din A (10 mgyml) alone or with nocodazole (20 mgyml) was added PM11, the 1.0-kb rat protease domain (PR) with PM8 and to labeling cell medium, and the cells were incubated in the PM11, the human kringle 1 domain (K1H) with PM15 and presence of vitamin K or warfarin for 24 h. PM3, the human kringle 2 domain (K2H) with PM14 and Enzymes and Reagents. Oligonucleotides were synthesized PM10, the rat kringle 1 domain (K1R) with PM13 and PM3, by Ransom Hill Bioscience (Ramona, CA). Restriction en- and the rat kringle 2 domain (K2R) with PM12 and PM9. These 1y2 zymes, T4 DNA ligase, and Wizard DNA maxiprep kits were PCR products were subcloned into pBluescript ( ), respec- obtained from Promega. Calf intestine alkaline phosphatase tively. PcDNA3-FIIHHR was generated by ligating the PGH was obtained from New England Biolabs. Mammalian expres- Bam yBbs Bbs insert ( HI I) and the KH insert ( I) with the PR sion vector pcDNA3 was purchased from Invitrogen. insert (BbsIyXbaI) into pcDNA3 (BamHIyXbaI). PcDNA3- The transformed human embryonic kidney cell line FII was generated by ligating the PG insert (BamHIy RRH R (HEK293) and the human hepatoma cell line (HepG2) were BbsI) and the K insert (BbsI) with the P insert (BbsIyXbaI) R H obtained from American Type Culture Collection. FBS was into pcDNA3 (BamHIyXbaI). PcDNA3-FII was generated HRH purchased from HyClone. [35S]methionine (43.5 TBqymmol) by ligating the PG insert (BamHIyBbsI) and the K insert H R was obtained from DuPontyNEN. Vitamin K was purchased (BbsI) with the P insert (BbsIyXbaI) into pcDNA3 (BamHIy H from Abbott. Warfarin was provided by the Wisconsin Alumni XbaI). PcDNA3-FIIRHR was generated by ligating the PGR y Research Foundation (Madison, WI). Kodak x-ray film was insert (BamHI BbsI) and the KH insert (BbsI) with the PR insert (BbsIyXbaI) into pcDNA3 (BamHIyXbaI). PcDNA3- purchased from Eastman Kodak. Fluoro-Hance solution was FII was generated by ligating the PG insert (BamHIy purchased from Research Products International.