Da-Yun Jin

Expression and Characterization of the Naturally Occurring Mutation L394R in Human γ-Glutamyl Carboxylase

Patients with mutation L394R in γ-glutamyl carboxylase have a severe bleeding disorder because of decreased biological activities of all vitamin K-dependent coagulation proteins. Vitamin K administration partially corrects this deficiency. To characterize L394R, we purified recombinant mutant L394R and wild-type carboxylase expressed in baculovirus-infected insect cells. By kinetic studies, we analyzed the catalytic activity of mutant L394R and its binding to factor IXs propeptide and vitamin KH2. Mutant L394R differs from its wild-type counterpart as follows: 1) 110-fold higher K i for Boc-mEEV, an active site-specific, competitive inhibitor of FLEEL; 2) 30-fold lower V max/K m toward the substrate FLEEL in the presence of the propeptide; 3) severely reduced activity toward FLEEL carboxylation in the absence of the propeptide; 4) 7-fold decreased affinity for the propeptide; 5) 9-fold higher K m for FIXproGla, a substrate containing the propeptide and the Gla domain of human factor IX; and 6) 5-fold higher K m for vitamin KH2. The primary defect in mutant L394R appears to be in its glutamate-binding site. To a lesser degree, the propeptide and KH2 binding properties are altered in the L394R mutant. Compared with its wild-type counterpart, the L394R mutant shows an augmented activation of FLEEL carboxylation by the propeptide.

Osteocalcin binds tightly to the gamma-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins.

Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site.

Evaluation of warfarin resistance using transcription activator-like effector nucleases-mediated vitamin K epoxide reductase knockout HEK293 cells

Single nucleotide polymorphisms in the vitamin K epoxide reductase (VKOR) gene have been successfully used for warfarin dosage prediction. However, warfarin resistance studies of naturally occurring VKOR mutants do not correlate with their clinical phenotype. This discrepancy presumably arises because the in vitro VKOR activity assay is performed under artificial conditions using the non‐physiological reductant dithiothreitol.

Transgene expression levels and kinetics determine risk of humoral immune response modeled in factor IX knockout and missense mutant mice

Immune responses leading to antibody-mediated elimination of the transgenic protein are a concern in gene replacement for congenital protein deficiencies, for which hemophilia is an important model. Although most hemophilia B patients have circulating non-functional but immunologically crossreactive factor IX (FIX) protein (CRM+ phenotype), inciting factors for FIX neutralizing antibody (inhibitor) development have been studied in crossreactive material-negative (CRM−) animal models. For this study, determinants of FIX inhibitor development were compared in hemophilia B mice, in which circulating FIX protein is absent (CRM− factor IX knockout (FIXKO) model) or present (CRM+ missense R333Q-hFIX model) modeling multiple potential therapies. The investigations compare for the first time different serotypes of adeno-associated virus (AAV) vectors (AAV2 and AAV1), each at multiple doses, in the setting of two different FIX mutations. The comparisons demonstrate in the FIXKO background (CRM− phenotype) that neither vector serotype nor vector particle number independently determine the inhibitor trigger, which is influenced primarily by the level and kinetics of transgene expression. In the CRM+ missense background, inhibitor development was never stimulated by AAV gene therapy or protein therapy, despite the persistence of lymphocytes capable of responding to FIX with non-inhibitory antibodies. This genotype/phenotype is strongly protective against antibody formation in response to FIX therapy.

Identification of a gene encoding a typical γ‐carboxyglutamic acid domain in the tunicate Halocynthia roretzi

Summary.  We report the identification of a gene capable of encoding a novel Gla (γ‐carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla‐RTK; it has a Gla domain typical of human vitamin K‐dependent coagulation factors, a transmembrane domain, and a receptor tyrosine kinase domain. The receptor tyrosine kinase domain is very similar to the ARK (adhesion‐related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c‐Mer. This gene also encodes a propeptide that binds to the human gamma‐glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla domain is highly homologous to the Gla domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase domain is highly homologous to Axl.

Characteristics of recombinant W501S mutated human γ‐glutamyl carboxylase

Summary.  A mutation (W501S) in the vitamin K‐dependent γ‐glutamyl carboxylase (VKC) that leads to a congenital bleeding disorder was recently discovered in two patients. To characterize the enzyme defect, recombinant VKC‐W501S was expressed in and purified from insect cells. The major effect of the mutation appears to be to decrease the affinity of the carboxylase for the propeptide of its substrates. This observation agrees with recent data that place part of the propeptide binding site within residues 495–513 of VKC. Additionally, we demonstrate that the affinity between descarboxy osteocalcin (d‐OC) and VKC remains unaffected by the W501S mutation. This confirms earlier data that the high‐affinity site for d‐OC is not located on the propeptide binding domain of VKC. Two properties of the enzyme suggest an explanation for the observation that vitamin K supplementation ameliorates the effects of the mutation: (i) since full carboxylation requires the propeptide to remain bound to the enzyme sufficiently long for full carboxylation, a reduced affinity can cause its premature release before carboxylation is complete; (ii) propeptide binding results in a decrease of the KM for vitamin K hydroquinone in wild‐type, but not in mutant carboxylase, resulting in increased vitamin K requirement of affected subjects.

Warfarin and vitamin K epoxide reductase: a molecular accounting for observed inhibition

Vitamin K epoxide reductase (VKOR), an endoplasmic reticulum membrane protein, is the key enzyme for vitamin K-dependent carboxylation, a posttranslational modification that is essential for the biological functions of coagulation factors. VKOR is the target of the most widely prescribed oral anticoagulant, warfarin. However, the topological structure of VKOR and the mechanism of warfarins inhibition of VKOR remain elusive. Additionally, it is not clear why warfarin-resistant VKOR mutations identified in patients significantly decrease warfarins binding affinity, but have only a minor effect on vitamin K binding. Here, we used immunofluorescence confocal imaging of VKOR in live mammalian cells and PEGylation of VKORs endogenous cytoplasmic-accessible cysteines in intact microsomes to probe the membrane topology of human VKOR. Our results show that the disputed loop sequence between the first and second transmembrane (TM) domain of VKOR is located in the cytoplasm, supporting a 3-TM topological structure of human VKOR. Using molecular dynamics (MD) simulations, a T-shaped stacking interaction between warfarin and tyrosine residue 139, within the proposed TY139A warfarin-binding motif, was observed. Furthermore, a reversible dynamic warfarin-binding pocket opening and conformational changes were observed when warfarin binds to VKOR. Several residues (Y25, A26, and Y139) were found essential for warfarin binding to VKOR by MD simulations, and these were confirmed by the functional study of VKOR and its mutants in their native milieu using a cell-based assay. Our findings provide new insights into the dynamics of the binding of warfarin to VKOR, as well as into warfarins mechanism of anticoagulation.