Protein Science (1997), 6185-196. Cambridge University Press. Printed in the USA. Copyright 0 1997 The Protein Society y-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function SHMUEL GILLIS,' BARBARA C. FURIE,' BRUCE FURIE,' HIMAKSHI PATEL: MICHAEL C. HUBERTY: MARY SWITZER: W. BARRY FOSTER: HUBERT A. SCOBLE: AND MICHAEL D. BOND* 'The Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center, Department of Medicine and Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 'Genetics Institute Inc., Andover, Massachusetts (RECEIVED September 9, 1996 ACCEP~EDOctober 16, 1996) Abstract The y-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IXIy40E is uncarboxylated at residue 40 and factor IX/y36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully y-carboxylated recombinant factor IX, unfractionated recombinant fac- tor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca(II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX speciesunderwent the Ca(I1)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 1251-labeledplasma-derived factor IX at concentrations of 2-6 nM. Func- tionally, factor IX/y36,40E and factor IXIy40E were similar to fully y-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IXly36,40E and factor IXIy40E. These results demonstrate that the y-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined. Keywords: calcium-binding protein; Gla; hemophilia B; vitamin K Factor IX is a vitamin K-dependent plasma zymogen that plays a function requires the presence of Gla residues that participate di- central role in blood coagulation. Patients deficient in factor IX rectly in Ca(I1) binding via their malonate side chains (Furie & activity have the bleeding disorder hemophilia B. In common with Furie, 1988; Freedman et al., 1995a). Gla is formed by a post- the other vitamin-K dependent blood coagulation proteins, fac- translational modification in which glutamic acid residues undergo tor IX contains an amino-terminal Gla domain that is responsible y-carboxylation mediated by the enzyme y-glutamyl carboxylase for its phospholipid binding properties (Furie & Furie, 1988). This (Furie & Furie, 1988). The Gla domains in the vitamin-K depen- dent proteins contain 9-12 Gla residues; of these, the N-terminal nine Gla residues are fully conserved and the tenth is highly con- Reprint requests to: BNce Furie* Division Of Hematology-oncology, served, Factor IX is unique in possessing two additional distal GIa New England Medical Center, 750 Washington St., Boston, Massachusetts 02111_-.._. residues at positions40 36 and (Vermeer, 1990). Abbreviurions: AAAS, aromaticamino acid stack; Achro-K, Achromo- The rnetal-free and calcium ion-bound structures of the fac- bacter Protease I; DHB, 2,5-dihy&oxybenzoic acid; dPE, dansyl-phospha- tor IX Gla and AAAS domains have been determined recently by tidylethanolamine; EGE epidermal growth factor; Gla, y-cahwglutamic NMR spectroscopy (Freedman et al., 1995a, 1995b). nemetal-free acid; HMB, 2-hydroxy-5-methoxybenzoicacid; MALDI-TOF MS, matrix assisted laser desorption ionization time-of-flight maSSspectromeny; PACE, structure is relatively disordered, whereas the calcium ion-bound paired basic amino acid cleaving enzyme; PC, phosphatidylcholine; PITC, form is substantially ordered, consisting of a large amino-terminal phenylisothiocyanate; PS, phosphatidylserine. loop (residues 1-12), three helical segments (residues 14-17,25-32, 185 S. Gillis et al. and 35-46), and a disulfide loop (residues 18-23). The three- dimensional structure of the polypeptide backbone of the calcium- bound form is very similar to that of prothrombin fragment 1 (Soriano-Garcia et al., 1992), but small differences in the N-terminal loop may befunctionally important. In the calcium ion-bound form, the amino-terminal nine Gla residues are oriented to the interior of the protein, consistent with an internal Ca(I1) binding pocket. Gla residues 33, 36, and 40 are on the solvent-exposed surface of the calcium ion-bound structure. Their role has not been defined. Gla 40 has been postulated to be required for the stabilization of the Gla domain carboxyl-terminal a-helix through ionic interactions (Freed- man et al., 1995a). Others have suggested, based on the X-ray struc- ture of the factor IX EGF-like domain, that Gla 40 may be a Ca(I1) binding site required for the correct folding of the EGF domain over the adjacent Gla domain (Rao et al., 1995). MINUTES Gla at residue 33 may be important for factor IX activity be- cause a mutation of the equivalent residue in prothrombin to as- Rec. Factor IX partic acid yielded aprotein with reduced coagulantactivity (Ratcliffe et al., 1993). A Gla to Asp mutation at residue 33 causes a severe deficiency in factor IX in a patient with hemophilia B; E however, the specific activity of this mutant is not known (Koeberl et al., 1989). The importance of Gla 36 and Gla 40 in factor IX N function remains unknown. Recombinant factor IX, in contrast to recombinant prothrombin (Jorgensen et al., 1987; Ratcliffe et al., 1993), has been found to be under-y-carboxylated, especially when expressed at high levels (Kaufman et al., 1986; Derian et al., 1989). In this report, we describe the isolation and purification of two partially y-carboxylated species of recombinant factor IX and demonstrate that they are 0 20 40 lacking y-carboxylation either at residue 40 or at both residues 36 MINUTES and 40. We have characterized these species functionally and com- pared them with plasma-derived human factor IX. We show that Gla 36 and Gla 40 are not required for any function of human 01 factor IX that was examined. Results Factor IX undergoes numerous posttranslational modifications, in- cluding the y-carboxylation of 12 glutamic acid residues in the Gla and AAAS domains (Furie & Furie, 1988; Vermeer, 1990), P-hydroxylation of Asp 64 (Femlund & Stenflo, 1983; Furie & Furie, 1988), and 0-glycosylation of two sites in the first EGF domain, at Ser 53 and Ser 61, the latter of which contains sialic 0 I acid (Bharadwaj et al., 1995). The activation peptide contains sev- 0 20 40 eral glycosylation, sulfation, and phosphorylation sites (Agarwala MINUTES et al., 1994; Bond et al., 1994a). In order to obtain species of Fig. 1. Mono Q HPLC separation of recombinant factor IX species. A: factor IX that lack Gla at specific residues, we fractionated recom- Recombinant factor IX samples were prepared in 20 mM Tris, pH 9.0, and binant factor IX expressed in Chinese hamster ovary cells. This injected in volumes of 0.1-2.0 mL. Elution employed a 0.3-0.4 M NaCl expression system yields nearly completely processed factor IX, gradient. Sample A represents results for a typical sample of recombinant factor IX. Sample B represents a sample with unusually high levels of but a component of recombinant factor IX is undercarboxylated peak 1. B: factor IX was analyzed by Mono Q chromatography as above, and thus served as a sourceof homogeneous partially carboxylated before and after removal of sialic acids with neuraminidase. C: Mono Q factor IX. chromatography before and after activation of recombinant factor IX with Application of unfractionated recombinant factor IX to a Mono Q factor XIa (1 :200, w/w) in 50 mM Tris, 0.15 M NaCI, 5 mM CaCI2, pH 7.5, ion exchange chromatography column yielded three major frac- for 1 h at 37°C. tions observed as peaks 1, 2, and 3. Peak 3 included a small shoul- der (Fig. 1A). To determine which modifications were responsible for the multiple species observed by ion exchange chromatography, tion differences. A sample of recombinant factor IX was also stud- a sample of recombinant factor IX was analyzed before and after ied before and after activation with factor XIa, a process that removes enzymatic desialylation. Desialylation induced a shift in retention the activation peptide (and any modifications therein). The chro- times, but did not change the chromatographic profile (Fig. 1B). matogram again demonstrated the same three major peaks, and there Therefore, the observed fractionation does not result from sialyla- was no shift in the retention times (Fig. IC). The results show that Gla 36 and Gla 40 of human factor TX 187 Table 1. Gla content of Mono Q purified recombinant factor IX speciesa 0.2 Species mol Gldmol protein Peak 1 10.3 -C 0.3 Peak 2 11.2 * 0.2 0 Peak 3 12.1 * 0.2 12-Gla Species Unfractionatedrecombinant factor IX 11.5 f 0.2 K3[6-Gla] Plasma-derived factor IX 12.0 * 0.3 Factor X 10.6 f 0.3 : 0.1 "Gla content was determined by amino acid analysis following alkaline v -0 hydrolysis as described in Materials and methods.The raw values of mol Glal N mol protein for each sample was normalized to yield mol Glahol ui 11-Gla Species 12.0 P protein for the plasma-derived factor included in each set of assays.