Toshizumi Tanabe

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product

Human single‐chain urokinase‐type plasminogen activator without an N‐glycosylation site (scu‐PA‐Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase‐type plasminogen activator (u‐PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N‐terminal amino acid sequence of major species was identical to that of the low molecular weight two‐chain u‐PA. Some approaches to minimizing the proteolysis of scu‐PA‐Q302 were attempted. Addition of Triton X‐100, l‐arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu‐PA‐Q302 and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A‐ or proteinase B‐deficient strains of yeast did not reduce the degradation. Co‐expression of scu‐PA‐Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis.

Effects of macrophage colony-stimulating factor (M-CSF) on lipopolysaccharide (LPS)-induced mediator production from monocytes in vitro.

M-CSF is a macrophage-lineage-specific growth factor that causes proliferation and differentiation of progenitor cells in the bone marrow. To investigate the effects of M-CSF on more matured cells, human monocytes were cultured in the presence or absence of M-CSF for 6 days. Addition of M-CSF at more than 10(2) U/ml resulted in higher viability and caused morphological differentiation to large macrophage-like cells. LPS-induced mediator production was also compared between M-CSF-treated and control cell. Monocytes were incubated with or without M-CSF for 3 days, and were stimulated with 1 microgram/ml of LPS for 2 days. IL-1 beta was not detected in the both culture supernatants, and PGE2 production was not influenced by M-CSF. However, amounts of G-CSF, GM-CSF, IL-6, and TNF-alpha produced in response to 1 microgram/ml of LPS were 1.5 to 2 times greater from monocytes treated with 10(4) U/ml of M-CSF than from control cells. The priming effect of M-CSF on LPS-induced cytokine production was found to require 3-day preincubation, and reached a maximum at the concentration of 10(4) U/ml. M-CSF-treated cells responded to a 10 times lower concentration of LPS than control cells in terms of cytokine production. M-CSF was also shown by flowcytometric analysis to influence the expression of CD14, a receptor for LPS, which might render monocytes more sensitive to LPS.

Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris.

To produce a thrombi-targeting plasminogen activator, we expressed a fused gene that contains a modified pre-sequence of Mucor pussilus rennin (MPR) followed by a chimeric gene of single-chain urokinase-type plasminogen activator (scu-PA)::annexin V (AV). The fused gene was ligated into an integrative vector, under the control of the alcohol oxidase 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transformants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per ml of culture medium under optimal conditions. It contained three tandem copies of the full-size vector disruptively integrated into the AOX1 sequence. Western blot analysis revealed that the secreted chimera was highly susceptible to proteolysis. Addition of excess amino acids (aa) to the culture medium minimized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was the intact form consisting of a single-chain and showing full enzyme activity after activation by plasmin. The latter was an enzymatically processed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calcium-dependent phospholipid (PL)-binding affinities similar to the parent AV.

Effects of macrophage colony-stimulating factor (M-CSF) on protease production from monocyte, macrophage and foam cell in vitro: a possible mechanism for anti-atherosclerotic effect of M-CSF.

M-CSF is a growth factor that stimulates proliferation and differentiation of monocyte/macrophage-lineage cells. In our previous studies, M-CSF regresses atherosclerotic lesions preformed in aorta of high cholesterol-fed rabbit. Immunohistochemical analysis indicated that extracellular matrix (ECM), such as collagen, was especially eliminated in the intima of atherosclerotic lesion. To define the collagen-lowering potential of M-CSF, we have studied the effects of M-CSF on production of collagen-degrading proteases, such as MMP-1, -9 and urokinase in vitro. Monocytes freshly isolated from human peripheral blood produced MMP-9, but not urokinase, and M-CSF enhanced MMP-9 production. Macrophages were prepared by culturing monocytes for 10 days in the presence or absence of M-CSF, and protease production was assayed. M-CSF augmented production of MMP-9 and urokinase in a dose-dependent manner. M-CSF also enhanced MMP-1 production of macrophages, but not significantly. Foam cells were prepared by culturing macrophages in the presence of acetyl LDL, and protease production from these cells were also elevated by M-CSF. These results suggest that M-CSF exogenously administered in atherosclerotic rabbits might regress the thickened intima by activating macrophages to degrade collagen accumulated in the lesion.

Effect of deletion of epidermal growth factor-like domain on plasma clearance of pro-urokinase

Abstract In order to study the relationship between the structure of pro-urokinase (pro-UK) and its clearance rate from circulation, three epidermal growth factor (EGF)-like domain deleted pro-UK mutants were constructed. Δ10–42, pro-UK, Δ10–45 pro-UK, and Δ10–49 pro-UK lacking Asn-10—Cys-42, Asn-10—Asp-45, and Asn-10—Thr-49, respectively, were compared with natural and recombinant pro-UKs (n-pro-UK and r-pro-UK) in terms of amidolytic activity and plasma clearance rate. Each pro-UK mutant gave almost the same enzymatic constants as n- and r-pro-UKs in terms of amidolytic activity. When 125I-labelled pro-UK and its derivatives were intravenously injected into rats, the clearance rate of pro-UK derivatives lacking EGF-like domain was significantly prolonged compared with those carrying intact EGF-like domain; for instance, the half-lives of Δ10–42 pro-UK, Δ10–45 pro-UK and Δ10–49 pro-UK were 8.2 ± 1.5 min, 6.3 ± 0.3 min and 7.1 ± 0.3 min, respectively, while those of n- and r-pro-UKs were 1.6 ± 0.1 min and 2.3 ± 0.0 min. Therefore, it is suggested that the pro-UK molecule is partially recognised and cleared by hepatic cells through EGF-like domain.

Macrophage colony-stimulating factor (M-CSF) augments cytokine induction by lipopolysaccharide (LPS)-stimulation and by bacterial infections in mice.

We studied the effects of M-CSF on cytokine induction in vivo by LPS or by bacterial infection by comparing between the serum cytokine levels of mice administered with and without M-CSF. M-CSF at 250 micrograms/kg/day for 3 days significantly augmented serum IL-6 level induced by a subsequent injection of 25 micrograms/kg of LPS. The augmented IL-6-induction was dose-dependent from 50 to 1250 micrograms/kg/day of M-CSF, and required 2- to 3-doses of M-CSF at 250 micrograms/kg/day. Mice primed with M-CSF induced IL-6 in response to a 5-fold lower dose of LPS, and also produced higher levels of IL-1 alpha, IL-10, GM-CSF, TNF-alpha, and IFN-gamma than control mice. The priming effect of M-CSF was transient and reversible, and elicited independently of T-cells. An injection with intact bacteria, such as Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus also induced IL-6 in normal mice, and M-CSF administration augmented the induction of these cytokines. These results showed that M-CSF positively regulates LPS-dependent and -independent cytokine induction, suggesting a defensive effect against infectious agents through enhanced cytokine production.

EFFECT OF RECOMBINANT HUMAN MACROPHAGE-COLONY STIMULATING FACTOR ON MARROW, SPLENIC, AND PERIPHERAL HEMATOPOIETIC PROGENITOR CELLS IN MICE

The effects of human macrophage colony‐stimulating factor (M‐CSF) on marrow, splenic, and peripheral progenitor cells (CFU‐M, CFU‐GM, and CFU‐G) were investigated in mice administered recombinant human M‐CSF (8‐4,000 μg/kg). Single injection of 4,000 μg/kg of M‐CSF resulted in a decrease in the number of marrow progenitor cells (CFU‐M, CFU‐GM, and CFU‐G) on day 2 followed by a gradual increase, returning to the original level on day 4 or 5. In contrast, each type of splenic progenitors tested for started to increase markedly on day 2, reaching a level 4‐ to 15‐fold higher than that of the basal value on day 3 or 4. Peripheral CFU‐M, CFU‐GM, and CFU‐G also increased on day 2. In addition, administration of 800 μg/kg of M‐CSF in mice caused a decrease in marrow CFU‐G, as well as an increase in splenic CFU‐G. The present results indicate that treatments of mice with pharmacological concentrations of human M‐CSF affect the number of progenitor cells not only of monocyte/macrophage lineage but also of granulocyte lineage. Also, the coincidence between decrease of marrow progenitor cells and increase of splenic and peripheral progenitor cells suggests that the progenitor cells are released from bone marrow to peripheral blood and reseeded to the spleen by the action of M‐CSF.

Intravenously administered macrophage colony-stimulating factor (M-CSF) specifically acts on the spleen, resulting in the increasing and activating spleen macrophages for cytokine production in mice

IL-6 was transiently expressed in sera of mice after a bolus intravenous injection with LPS and it peaked 2 h later. Intravenous administration of M-CSF at 250 micrograms/kg/day for 5 days prior to an injection of 25 micrograms/kg of LPS elevated the serum IL-6 level 10-fold higher than that of mice which were not given M-CSF. Although M-CSF had no effect on the number of macrophages in alveoli and peritoneal cavity, it tripled the number of spleen macrophages and increased macrophage-progenitor cells 7-fold when injected intravenously. Spleen macrophages from M-CSF-injected mice produced 5-fold more IL-6 in response to LPS-stimulation in-vitro. However, M-CSF-injection had lesser effects on LPS-induced IL-6 production from liver, alveolar and peritoneal macrophages. Exogenously administered M-CSF was detected at higher concentration and for longer duration in the spleen than in any other organs examined. Spleen macrophages incubated in-vitro with more than 1000 U/ml of M-CSF for 3 days also produced more LPS-induced IL-6 than untreated cells. These results indicate that intravenously administered M-CSF not only enhances macrophage development in the spleen, but also primes mature macrophages for cytokine production.

Expression of c-fms Proto-Oncogene Product by Ovarian Cancer Cell Lines with Effects of Macrophage Colony-Stimulating Factor on Proliferation

We studied the production of macrophage colony-stimulating factor (M-CSF) and the expression of c-fms mRNA, an M-CSF receptor, in four human ovarian cancer cell lines. All four cell lines expressed c-fms mRNA while three secreted M-CSF into the culture medium. The exogenous administration of M-CSF caused no significant enhancement of cellular proliferation in any cell line. Interestingly, the proliferation of KK cells was not affected by anti-M-CSF antibody. These results, taken together with the fact that ovarian cancer cells simultaneously produce M-CSF and c-fms, suggest that an autocrine mechanism may modulate cellular proliferation.

Pharmacokinetics and biochemical properties of human pro-urokinase variants carrying the deletion in epidermal growth factor-like domain

Abstract Two variants of pro-urokinase (pro-UK), Δ11–32pro-UK lacking the first and the second loops (Cys11Asn32) within the EGF-like domain and δ33–42pro-UK lacking the third loop (Cys33Cys42), were prod in Chinese hamster ovary cells. They were compared with natural pro-UK (n-pro-UK) and Δ10–42pro-UK lacking entire EGF-like domain (Hiramatsu et al1) in terms of clearance rate and biochemical properties. Fibrinolytic activity half-lives of these mutants in rat circulation after bolus administration were 7.1 min for Δ11–32pro-UK and 6.8 min for Δ33–42pro-UK, which were about 3.5 times longer than that of n-pro-UK (2.0 min) and slightly shorter than that of Δ10–42pro-UK (7.6 min). The amidolytic activity of these deletion mutants were almost the same as n-pro-UK in terms of both Km and kcat (3.4–4.O × 10−4 M, and 8.4–10.O × 103 min−1 respectively), suggesting that the protease domain was not affected by the deletion in EGF-like domain. It was also found that the binding ability to fibrin-celite was decreased by the absence of the third loop of the EGF-like domain. In addition, conversion from the single chain form to the two chain form by the action of plasmin was slowed in proportion to the size of the deletion in the EGF-like domain.