Jpn. J. Pharmacol. 90, 210 – 213 (2002)
FORUM MINIREVIEW Development and Application of Chymase Inhibitors Recombinant Human Chymase Produced by Silkworm-Baculovirus Expression System: Its Application for a Chymase Detection Kit Takeo Suzuki1,*, Hiroki Kaki1, Shinichi Naya1, Soji Murayama1, Akira Tatsui2, Akihiko Nagai2, Shinji Takai3 and Mizuo Miyazaki3 1
Chuo-Sanken Laboratory, Katakura Industries Co., Ltd., Saitama 350-1332, Japan 2 Fukushima Research Laboratories, Toa Eiyo, Ltd., Fukushima 960-0280, Japan 3 Department of Pharmacology, Osaka Medical College, Osaka 569-8686, Japan Received July 25, 2002 Accepted August 30, 2002
ABSTRACT—Human chymase is a mast cell-derived serine proteinase, which is a non-angiotensin converting enzyme angiotensin II-generating enzyme. It appears to participate in various diseases, but it is unclear whether chymase plays major roles in physiological and pathophysiological functions in vivo. To obtain information on the physiological and pathophysiological functions of chymase and to search for diseases in which chymase participates, in the present study, we aimed at producing recombinant human chymase in large quantities and at developing an ELISA system using anti-human chymase antibodies. A recombinant human chymase was produced by a silkworm-baculovirus expression system. The recombinant chymase in active form was efficiently purified from larval hemolymph using cation-exchange and heparin column chromatography. This recombinant enzyme was enzymatically identical with native human chymase. On the other hand, the stability of the recombinant enzyme in cultured medium for mammalian cells at 37°C was very high as compared with the stability of the native enzyme; 20% of the activity was maintained 120 h after addition of medium. These results indicated that the recombinant enzyme could also utilize in vitro and in vivo assay systems. We obtained several anti-chymase monoclonal antibodies by using the recombinant human chymase as antigen. These antibodies were used to construct an ELISA system for measuring the chymase concentration in blood. As a result of preliminary examination using this ELISA system, it was shown that the chymase concentration in each serum from hypertensive patients is significantly higher than in normal serum. The ELISA system will be applicable for clinical diagnosis and in vivo evaluation systems for chymase-targeting drugs. Keywords: Recombinant chymase, Silkworm-baculovirus expression system, Assay, ELISA, Physiological function
among species. In humans, chymase participates to a greater extent than angiotensin converting enzyme (ACE) in the Ang II-forming pathway. For example, Urata et al. has reported that 80% of the Ang II-generation ability was occupied by chymase in heart (4). It has also been found that human chymase is involved in the restricted cleavage of big-endothelin (5, 6), and in the activation of collagenases (7) or cytokines (8). However, the original physiological functions are not yet fully clarified. On the other hand, it is supposed that chymase participates in various diseases because chymase expression is accelerated in local tissues in cases of cardiovascular diseases and similar disorders. However,
Human chymase is a mast cell-derived chymotrypsinlike-proteinase. It has been characterized as an angiotensin II (Ang II) generating enzyme acting on angiotensin I (Ang I) as the physiological substrate. Chymases are known to cleave Ang I at different sites in different species. For example, human, monkey and hamster chymases cleave the Phe8-His9 bond of Ang I, whereas rat chymase cleaves predominantly the Tyr4-Lle5 bond of Ang I to form inactive fragments (1 – 3). Therefore, it is anticipated that the physiological functions of chymase differ greatly *Corresponding author. FAX: +81-42-954-2172 E-mail:
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Recombinant Human Chymase
the pathophysiological functions are also not yet understood. To understand the physiological and pathophysiological functions of human chymase, in this study, we aimed at producing a recombinant human chymase in large quantities and at developing an ELISA system using anti-human chymase antibodies. Production of the recombinant human chymase We succeeded in obtaining recombinant human chymase in large quantities by using a silkworm-baculovirus expression system. The silkworm-baculovirus expression system is a very efficient protein production system. To prevent auto-degradation during expression and in the purification process, we took the strategy of expressing human chymase as an inactive form with an enterokinase cleavage sequence (DDDDK) at the N-terminal. After purification, the enzyme was activated by enterokinase. Human chymase cDNA was cloned using primers with an enterokinase cleavage sequence by the reverse transcription-polymerase chain reaction (RT-PCR) method. The cloned chymase cDNA containing the enterokinase cleavage sequence at the N-terminal (EK+chymase) was inserted into a transfer vector (pYNGsig) with the secretory signal from the major hemolymph protein (30KP) in silkworm. The transfer vector with EK+chymase cDNA
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and baculovirus (CPd strain) genomic DNA were co-transfected into BmN cells (9). Then, the recombinant baculovirus was screened. When the lysates of cultured cells infected with each screened recombinant virus were reacted with enterokinase, the chymotrypsin activity in the lysates was specifically increased. Furthermore, the EK+chymase was efficiently secreted into the hemolymph from the recombinant virus infected-silkworm larvae. Purification and activation of the recombinant chymase The EK+chymase in the hemolymph was adsorbed on a cation-exchange column (SP-Toyopearl; Tosoh Co., Tokyo) and eluted by 50 mM phosphate buffer containing 0.5 M NaCl. Next, the eluate was adsorbed on a heparin affinity column (HiTrap Heparin; Amersham Biosiences, Uppsala, Sweden) and eluted by a 0.4 – 0.5 M NaCl linear gradient. Thus, 3.2 mg of the purified EK+chymase (electrophoretically single) were obtained from 100 ml of silkworm hemolymph (Fig. 1A). The purified EK+chymase was activated by enterokinase: 45 units of enterokinase was added to 1 m g of EK+chymase and incubated for 20 h at 25°C. Consequently, the reaction solution was purified again by heparin column chromatography, and the purified recombinant chymase in active form (mature-type) was obtained.
Fig. 1. Purification and activation of the recombinant chymase. A: SDS-PAGE (silver staining) analysis at each purification step of recombinant chymase (enterokinease recognition peptide fused at N-terminal: EK+chymase) produced by silkwormbaculovirus expression system. B: Activation of the EK+chymase by enterokinase. Mr: molecular weight marker.
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Fig. 2. Stability of the recombinant chymase and native chymase in BSA solution (1 mg/mL) or mammalian cultured cell medium (MEM containing 5% FBS) at 37°C. Closed square: recombinant chymase/BSA solution, open square: native chymase /BSA solution, closed circle: recombinant chymase / cultured cell medium, open circle: native chymase /cultured cell medium.
Characterization of the recombinant chymase The hydrolyzed activity (Km: 53.7 mM, kcat: 78.7 /s) of the activated recombinant chymase with Ang I as a substrate was nearly identical with the activity (Km: 67 mM, kcat: 65 /s) of the native human chymase (10). The substrate specificity of recombinant chymase was also similar to that of native chymase. Namely, the recombinant enzyme cleaved efficiently Ang I, neurotensin and big-endothelin-
1, whereas it could not cleave Ang II, substance P or bradykinin. Moreover, the recombinant enzyme was strongly inhibited by chymostatin and alpha-antitrypsin, but was not inhibited by leupeptin and aprotinin. Thus, its inhibitory profile was very similar to that of the native enzyme. On the other hand, the stability of the recombinant enzyme was significantly higher than that of the native enzyme. In particular, in cultured medium for mammalian cells at 37°C, 20% of the activity of the recombinant chymase was maintained 120 h after addition of medium, whereas the activity of native chymase rapidly decreased after several hours (Fig. 2). It is presumed that these differences in stability are caused by the variations of glycosylation of the two enzymes. Preparation of anti-chymase monoclonal antibodies We obtained several anti-chymase monoclonal antibodies by immunizing mice with intact recombinant chymase or chymase fragmented by lysylendopeptidase as the antigen. Because two of these antibodies (6D, 2D11G10D) were obviously different in the reaction pattern for lysylendopeptidase-cleaved chymase, it was considered that each antibody recognized a different antigen region. Moreover, it was shown that the 2D11G10D antibody could be stained immunohistochemically on specific mast cells expressing chymase using formalin-fixed sections with microwave treatment.
Fig. 3. Measurement of chymase concentration in the serum from renal hypertensive patients using the ELISA system. Each serum was diluted in 50 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 0.1% SDS, and heated at 100°C for 3 min. The treated each serum was applied on 96-well plate, which was immobilized by anti-chymase monoclonal antibody (6D), and incubated at 37°C for 60 min. After washing, the plate was reacted with biotin labeled antichymase monoclonal antibody (2D11G10D). After washing, the each well was colored with HRP labeled streptavidin, and then the absorbance was measured at 450 – 660 nm.
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Construction of ELISA system By using these antibodies, we tried to construct an ELISA system for measuring the chymase concentration in blood. The 6D antibody was coated on a 96-well plate, after blocking, and purified human vascular derived-chymase was reacted in each well. Next, the plate was reacted with the biotin-labeled 2D11G10D antibody, and each well was colored with horseradish peroxidase (HRP)-labeled streptavidin. Accordingly, it was clear that the measurement sensitivity was greatly increased when the sample was heated at 100°C for 3 min in 0.1% SDS-containing buffer. Furthermore, the concentration of chymase in hypertensive patient’s serum samples was actually measured by this ELISA system. As a result of preliminary examination using this ELISA system, it was shown that the chymase concentration in each serum from hypertensive patients is significantly higher than that in normal serum (Fig. 3). This result suggested that the concentration of chymase in serum might actually be accelerating along with the chymaserelated disease. Conclusion We succeeded in efficiently producing recombinant human chymase by using a silkworm-baculovirus expression system. This recombinant enzyme is especially stable in comparison with the native enzyme. This result indicated that the recombinant chymase could not only be used as a system for screening inhibitors, but also could be applied to various in vitro and in vivo assay systems. We obtained highly specific monoclonal antibodies and constructed an ELISA system for measurement of the chymase concentration in serum. By using this ELISA system, we are trying to elucidate the relationship between chymase concentration in serum and each disease. Consequently, we expect that the detailed or new pathological function of human chymase will be revealed, and this ELISA system will be applied to clinical diagnosis from now on.
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