[Gla-containing proteins].

To date, 16 Gla-containing proteins have been discovered in humans, 7 and 9 of which are involved or not-involved in the blood coagulation cascade, respectively. They have a common feature that the carbon at the γ-position of glutamic acid in the specific amino acid sequence of the protein molecule is carboxylated. γ-Glutamyl carboxylase catalyzes this reaction in the presence of vitamin K as a cofactor. Similar to phosphorylation and glycosylation, γ-carboxylation of the glutamic acid has been thought to be one of the post-translational modification for the activation of proteins. However, undercarboxylated, but not highly carboxylated osteocalcin has been found to exhibit regulatory activities of glucose metabolism and insulin sensitivity in mice, suggesting that there would be more comprehensive mechanisms in the regulation of protein functions by the carboxylation of glutamic acid and the decarboxylation of γ-glutamic acid.

Differential expression of benzophenone synthase and chalcone synthase in Hypericum sampsonii.

cDNAs encoding Hypericum sampsonii benzophenone synthase (HsBPS) and chalcone synthase (HsCHS) were isolated and functionally characterized. Differential expressions of HsBPS and HsCHS were monitored using quantitative polymerase chain reaction (PCR). In the vegetative stage, HsBPS was highly expressed in the roots; its transcript level was approx. 100 times higher than that of HsCHS. Relatively high transcript amounts of HsBPS were also detected in older leaves, whereas the youngest leaves contained higher transcript amounts of HsCHS. In the reproductive stage, maximum HsCHS expression was detected in flowers, the transcript level being approx. 5 times higher than that of HsBPS. The inversed situation with a 10-fold difference in the expression levels was observed with fruits. High transcript amounts for both proteins were found in roots.

High-level expression of functional recombinant human coagulation factor VII in insect cells.

Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human gamma-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future.

Activation of cancer cell migration and invasion by ectopic synthesis of coagulation factor VII.

Blood coagulation factor VII (fVII) is physiologically synthesized in the liver and released into the blood. Binding of fVII to tissue factor (TF) at sites of vascular injury triggers coagulation and hemostasis. TF/fVIIa complex formation on the surface of cancer cells plays important roles in cancer biology. Although fVII is synthesized by hepatocellular carcinoma, it remained unclear how TF/fVIIa complex formation and promigratory signaling can occur for most other cancers in extravascular locations. Here, we show by reverse transcription-PCR analysis that nonhepatic cancer cell lines constitutively express fVII mRNA and that endogenously synthesized fVIIa triggers coagulation activation on these cells. fVIIa expression in cancer cells is inducible under hypoxic conditions and hypoxia-inducible factor-2 alpha bound the promoter region of the FVII gene in chromatin immunoprecipitation analyses. Constitutive fVII expression in an ovarian cancer cell line enhanced both migration and invasion. Enhanced motility was blocked by anti-TF antibodies, factor Xa inhibition, and anti-protease-activated receptor-1 antibody treatment, confirming that TF/fVIIa stimulated migration by triggering cell signaling. This study shows that ectopic synthesis of fVII by cancer cells is sufficient to support proinvasive factor Xa-mediated protease-activated receptor-1 signaling and that this pathway is inducible under hypoxia.

Effects of resistance exercise and growth hormone administration at low doses on lipid metabolism in middle-aged female rats.

hGH (human growth hormone) is a key factor for metabolism as well as for growth. Lack of hGH usually increases LDL (low-density lipoprotein) while impairing exercise capacity and cardiac function [Amato G., Carella C., Fazio S., La Montagna G., Cittadini A., Sabatini D., Marciano-Mone C., Sacca L., and Bellastella A., 1993. Body composition, bone metabolism, and heart structure and function in growth hormone (GH)-deficient adults before and after GH replacement therapy at low doses. J. Clin. Endocrinol. Metab. 77, 1671-1676.]. It is still unclear, however, whether the administration of hGH to humans or animals has a synergic effects on body composition and the desired metabolic parameters with endurance resistance exercise. Therefore, the present study seeks to establish whether or not lipid metabolism is altered by both recombinant GH (growth hormone) and X (resistance exercise) in middle-aged female rats. GH administration resulted in greater weight gain compared with control and exercised animals, but the effect was reduced when combined with exercise. The increased body weight was largely due to increased muscle mass. Exercise did not result in any additional effect of GH on muscle mass. In the exercise group, hepatic HDL (high density lipoprotein) increased compared to the C (control group). The GX (growth hormone+exercise) group's serum and X group's kidney IGF-I (Insulin-like growth factor-I) concentration increased compared to the C group. In G and GX groups, serum insulin and leptin concentrations were higher than in the control, suggesting that GH may induce an insulin resistant state. Any gains in muscle mass were minimal and were not synergistic with exercise. These results suggest that hGH may not be useful for increasing performance in athletes and may induce and acquired insulin resistance.

Carboxylase overexpression effects full carboxylation but poor release and secretion of factor IX: implications for the release of vitamin K-dependent proteins.

Vitamin K-dependent (VKD) proteins are modified by the VKD carboxylase as they transit through the endoplasmic reticulum. In a reaction required for their activity, clusters of Glu's are converted to Gla's, and fully carboxylated VKD proteins are normally secreted. In mammalian cell lines expressing high levels of r-VKD proteins, however, under- and uncarboxylated VKD forms are observed. Overexpression of r-carboxylase does not improve carboxylation, but the lack of effect is not understood, and the intracellular events that occur during VKD protein carboxylation have not been investigated. We analyzed carboxylation in 293- and BHK cell lines expressing r-factor IX (fIX) and endogenous carboxylase or overexpressed r-carboxylase. The fIX secreted from the four cell lines was highly carboxylated, indicating fIX-carboxylase engagement during intracellular trafficking. The r-carboxylase was functional for carboxylation: overexpression resulted in a proportional increase in fIX-carboxylase complexes that yielded full fIX carboxylation. Interestingly, the carboxylated fIX product was not efficiently released from the carboxylase in r-fIX/r-carboxylase cells, resulting in decreased fIX secretion. r-Carboxylase overexpression changed the ratios of intracellular fIX to carboxylase, and we therefore developed an in vitro assay to test whether fIX levels affect release. FIX-carboxylase complexes were in vitro carboxylated with or without excess VKD substrate or propeptide. These analyses are the first to dissect the rates of release versus carboxylation and showed that release was much slower than carboxylation. In the absence of excess VKD substrate/propeptide, fIX in the fIX-carboxylase complex was fully carboxylated by 10 min, but 95% was still complexed with carboxylase after 30 min. The presence of excess VKD substrate/propeptide, however, led to a significant increase in VKD product release, possibly through a second propeptide binding site in the carboxylase. The intracellular analyses also showed that the fIX carboxylation rate was slow in vivo and was similar in r-fIX versus r-fIX/r-carboxylase cells, despite the large differences in carboxylase levels. The results suggest that the vitamin K cofactor may be limiting for carboxylation in the cell lines.

Expression and characterization of recombinant vitamin K-dependent gamma-glutamyl carboxylase from an invertebrate, Conus textile.

The marine snail Conus is the sole invertebrate wherein both the vitamin K-dependent carboxylase and its product, gamma-carboxyglutamic acid, have been identified. To examine its biosynthesis of gamma-carboxyglutamic acid, we studied the carboxylase from Conus venom ducts. The carboxylase cDNA from Conus textile has an ORF that encodes a 811-amino-acid protein which exhibits sequence similarity to the vertebrate carboxylases, with 41% identity and approximately 60% sequence similarity to the bovine carboxylase. Expression of this cDNA in COS cells or insect cells yielded vitamin K-dependent carboxylase activity and vitamin K-dependent epoxidase activity. The recombinant carboxylase has a molecular mass of approximately 130 kDa. The recombinant Conus carboxylase carboxylated Phe-Leu-Glu-Glu-Leu and the 28-residue peptides based on residues -18 to +10 of human proprothrombin and proFactor IX with Km values of 420 micro m, 1.7 micro m and 6 micro m, respectively; the Km for vitamin K is 52 micro m. The Km values for peptides based on the sequence of the conotoxin epsilon-TxIX and two precursor analogs containing 12 or 29 amino acids of the propeptide region are 565 micro m, 75 micro m and 74 micro m, respectively. The recombinant Conus carboxylase, in the absence of endogenous substrates, is stimulated up to fivefold by vertebrate propeptides but not by Conus propeptides. These results suggest two propeptide-binding sites in the carboxylase, one that binds the Conus and vertebrate propeptides and is required for substrate binding, and the other that binds only the vertebrate propeptide and is required for enzyme stimulation. The marked functional and structural similarities between the Conus carboxylase and vertebrate vitamin K-dependent gamma-carboxylases argue for conservation of a vitamin K-dependent carboxylase across animal species and the importance of gamma-carboxyglutamic acid synthesis in diverse biological systems.

Functional characterization of PCCA mutations causing propionic acidemia.

Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric enzyme composed of alpha and beta subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (alpha subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype-phenotype correlations in PCCA-deficient PA patients.

Vitamin K uptake in hepatocytes and hepatoma cells.

Hepatocellular carcinoma (HCC) or hepatoma cells have impaired ability to perform vitamin K-dependent carboxylation reactions. Vitamin K can also inhibit growth of HCC cells in vitro. Both carboxylation and growth inhibition are vitamin K dose dependent. We used rat hepatocytes, a vitamin K-growth sensitive (MH7777) and a vitamin K-growth resistant (H4IIE) rat hepatoma cell line to examine vitamin K uptake and vitamin K-mediated microsomal carboxylation. We found that vitamin K is taken up by normal rat hepatocytes against a saturable concentration gradient. The relative rates of uptake by rat hepatocytes and the two rat cell lines MH7777 and H4IIE correlated with their sensitivity to vitamin K-mediated cell growth inhibition. Pooled hepatocytes from liver nodules from rats treated with the hepatocarcinogen diethylnitrosamine (DEN) also had a reduced rate of vitamin K uptake. However, using a cell-free system, microsomes from both normal rat hepatocytes and the two rat hepatoma cell lines had a similar ability to support carboxylation mediated by exogenously added vitamin K. The results support the hypothesis that different sensitivity of hepatoma cells to vitamin K may be due to differences in vitamin K uptake and may be unrelated to the actions of vitamin K on carboxylation.

Propionic acidemia: analysis of mutant propionyl-CoA carboxylase enzymes expressed in Escherichia coli.

Deficiency of propionyl-CoA carboxylase (PCC) results in propionic acidemia, an autosomal recessive disorder characterized by ketoacidosis sufficiently severe to cause neonatal death. PCC is involved in the catabolism of branched-chain amino acids, odd-chain fatty acids, and cholesterol. The enzyme is a biotin-dependent mitochondrial protein composed of two heterologous subunits arranged into an 800-kDa alpha(6 )beta(6) dodecameric structure. Approximately 60 mutations have been reported in the nuclear genes PCCA and PCCB that encode the two PCC subunits. The vast majority of these mutations have not been examined at the protein level. We present an initial characterization of 13 mutations located in exons 1, 3-7, and 12-14 of PCCB. After expression in E. coli, these recombinant mutant enzymes were analyzed for stability, biotinylation, alpha-beta subunit interaction, and activity. Our results show a functional dichotomy in these PCCB mutations with some mutants (R44P, S106R, G131R, G198D, V205D, I408del, and M442T) capable of varying degrees of assembly but forming catalytically inactive PCC proteins. Other PCCB mutants (R165W, E168K, D178H, P228L, and R410W) that are PCC deficient in patient-derived fibroblasts, were found to be capable of expressing wild-type level PCC activity when assembled in our chaperone-assisted E. coli expression system. This result indicates that these mutations exert their pathogenic effect due to an inability to assemble correctly in patients' cells. This initial screen has identified a range of mutant PCC proteins that are sufficiently stable to be purified and subsequently used for structure-function analysis to further elucidate the complex relationship between genotype and phenotype in propionic acidemia.

Identification of the five hydrophilic residues (Lys-217, Lys-218, Arg-359, His-360, and Arg-513) essential for the structure and activity of vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase catalyzes the posttranslational conversion of glutamic acid to gamma-carboxyglutamic acid in vitamin K-dependent proteins. The clustered charged-to-alanine scanning mutagenesis of bovine carboxylase has identified five distinct candidate regions (I. Sugiura et al., J. Biol. Chem. 271, 17837-17844, 1996) with significant loss-of-function phenotype. To further specify the residues essential for the structure and function of the enzyme, Lys-217, Lys-218, Arg-359, His-360, Lys-361, Arg-513, and Lys-515 were analyzed by substituting to alanine individually. All the mutants except for K217A were expressed in Chinese hamster ovary cells. The carboxylase activities of R359A, H360A, and R513A decreased in parallel with the vitamin K epoxidase activities. Both carboxylations by R359A and H360A were stimulated saturatively at 1 microM factor IX propeptide (proFIX18) concentration, but that by R513A was not at a concentration up to 128 microM. K218A completely lost the enzyme activities but it cross-linked to the propeptide, suggesting that Lys-218 is critical for enzyme activity without affecting propeptide binding. We conclude that Lys-218, Arg-359, and His-360 are involved in the catalytic event, and Arg-513 participates in propeptide binding.

Cloning, structural organization, and transcriptional activity of the rat vitamin K-dependent gamma-glutamyl carboxylase gene.

The vitamin K-dependent gamma-glutamyl carboxylase gene was cloned from a rat liver genomic DNA library and the structural organization of this gene was determined. The carboxylase gene is 16.3 kb in length and contains 15 exons and 14 introns. DNA sequence analysis revealed that all 14 introns were U2-Type GT-AG introns. A 2.8-kb DNA fragment corresponding to the 5'-flanking region of the cloned gene demonstrated transcriptional activity in a rat liver cell line that is known to express the endogenous carboxylase gene. DNA sequence analysis of the proximal 331 bp of this 5'-flanking sequence reveals the absence of an identifiable TATA box. Consensus sequences for several transcription factors that may be important in regulating its tissue specific expression were identified. The isolation and characterization of the rat carboxylase gene provides essential information for the analysis of its regulation in vitro and in transgenic animal models.