High-level expression and preparation of recombinant human fibrinogen as biopharmaceuticals.

Fibrinogen is a large and complex glycoprotein containing two sets of each of three different chains (α, ß and γ). There have been no reports of high-level expression of fibrinogen at commercial levels using mammalian cultured cells such as CHO cells because of the difficulty in highly expressing a protein with such a complex structure. We achieved high-level (1.3 g/l or higher) expression of recombinant human fibrinogen using CHO DG44 cells by optimizing the expression system and culture conditions. We also succeeded in establishing a high-recovery preparation method for recombinant fibrinogen that rarely yields degraded products. To characterize the properties of the recombinant human fibrinogen, we performed SDS-PAGE; western blotting of the α, ß and γ chains using specific antibodies and scanning electron microscopy observations of fibrin fibres. We also evaluated the functional equivalence between recombinant fibrinogen and plasma fibrinogen with respect to the release of fibrinopeptides initiated by thrombin and its cross-linking properties. The basic properties of recombinant fibrinogen showed no apparent differences from those of plasma fibrinogen. Here, we report the development of methods for the culture and preparation of recombinant human fibrinogen of satisfactory quality that can be scaled up to the commercial level.

High-yield preparation of recombinant human α-thrombin for therapeutic use.

In this study, we established stable cell lines producing 1.5 mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200 µg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical.

Effects of selenoprotein P on the contraction and relaxation of the airway smooth muscle.

Selenoprotein P (SeP) not only represents the major selenoprotein in plasma, but also provides more than 50% of the total plasma selenium. However, there is no report concerning the direct action of selenium or selenium-containing compounds on the contraction and relaxation of the airway smooth muscle. Therefore, we investigated the effects of SeP and sodium selenite (SS) on the indirectly induced contraction and relaxation of the cat bronchi, and gel contraction of cultured bovine tracheal smooth muscle cells (BTSMC) induced by ATP. In the present results, SeP or SS suppressed the amplitude of twitch-like contractions of cat bronchiole without affecting the non-adrenergic and non-cholinergic (NANC) relaxations evoked by electrical field stimulation. SeP also suppressed the ATP-induced gel contraction of BTSMC. These results suggest that SeP suppresses the amplitude of twitch-like contraction of cat bronchiole by acting directly on the bronchiolar smooth muscle.

Selenoprotein P controls oxidative stress in cornea.

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.

Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood.

ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.

Serum selenoprotein-P levels in patients with inflammatory bowel disease.

OBJECTIVE: Selenoprotein-P is a selenium-rich serum protein that carries more than 50% of serum selenium. We evaluated changes in serum selenoprotein-P levels in patients with inflammatory bowel disease. METHODS: Serum selenoprotein-P levels were measured by enzyme-linked immunosorbent assay. Twenty healthy individuals (controls), 34 patients with ulcerative colitis, and 37 patients with Crohn's disease (CD) were studied. RESULTS: A highly significant correlation was found between the serum selenium and selenoprotein-P levels. There was no significant difference in serum selenoprotein-P levels between healthy controls (average 3.4+/-0.8 microg/mL, n=20) and patients with ulcerative colitis (3.0+/-1.0 microg/mL, n=34). Serum selenoprotein-P levels were significantly lower in patients with CD (average 1.8+/-0.5 microg/mL, n=37). Serum selenoprotein-P levels were significantly lower in the elemental diet group of patients who had CD (average 1.4+/-0.4 microg/mL, n=17) than in the non-elemental diet group of patients who had CD (average 2.1+/-0.3 microg/mL, n=20). CONCLUSION: We found that the serum selenoprotein-P level is decreased in patients with CD. It may be a useful marker to monitor the systemic selenium status in various disorders.

Domain structure of bi-functional selenoprotein P.

Human selenoprotein P (SeP), a selenium-rich plasma glycoprotein, is presumed to contain ten selenocysteine residues; one of which is located at the 40th residue in the N-terminal region and the remaining nine localized in the C-terminal third part. We have shown that SeP not only catalyses the reduction of phosphatidylcholine hydroperoxide by glutathione [Saito, Hayashi, Tanaka, Watanabe, Suzuki, Saito and Takahashi (1999) J. Biol. Chem. 274, 2866-2871], but also supplies its selenium to proliferating cells [Saito and Takahashi (2002) Eur. J. Biochem. 269, 5746-5751]. Treatment of SeP with plasma kallikrein resulted in a sequential limited proteolysis (Arg-235-Gln-236 and Arg-242-Asp-243). The N-terminal (residues 1-235) and C-terminal (residues 243-361) fragments exhibited enzyme activity and selenium-supply activity respectively. These results confirm that SeP is a bi-functional protein and suggest that the first selenocysteine residue is the active site of the enzyme and the remaining nine residues function as a selenium supplier.

Identification of selenoprotein P fragments as a cell-death inhibitory factor.

Megakaryoblastoma (Dami cells) cultured in a serum-free medium containing albumin, proliferated for three days but died on the fourth day. This cell death was not observed when human plasma was added, suggesting that human plasma contains a cell-death inhibitory factor. In order to identify this factor, we purified it from human plasma. N-terminal amino acid sequence analysis revealed that this factor is a mixture of C-terminal fragments of selenoprotein P, a major selenocysteine-containing protein in plasma. The specific activity (unit per pmol of selenium) of selenoprotein P fragments protein was 15-fold and 1900-fold higher than that of the full-length SeP and sodium selenite, respectively.