J. Biochem. 112, 476-481 (1992)
Preparation and Properties of Monoclonal Antibodies against Lipopolysaccharide-Sensitive Serine Protease Zymogen, Factor C, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes1 Yoshiki Miura,* Fuminori Tokunaga/' 2 Toshiyuki Miyata,**-3 Mateuko Moriyasu,"* Katsutoshi Yoshikawa,*** and Sadaaki Iwanaga**
Received for publication, April 13, 1992
Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and a-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not a-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Ka) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9X10", 0.6x10'°, and 1.8x10'° M, respectively. By using 2C12 and polyclonal antibody against factor C, an enzyme-linked immunosorbent assay for quantitative determination of factor C was established.
Horseshoe crab hemocytes are highly sensitive to Gramnegative bacterial endotoxins, lipopolysaccharides (LPS) (1-3). Exposure of the hemocytes to LPS results in the activation of the intracellular coagulation system, which involves at least three serine protease zymogens named proclotting enzyme (4), factor B (5), and factor C (6-12). The initial activator of the coagulation cascade, factor C, is a zymogen consisting of a single-chain glycoprotein with a molecular weight of 123,000. During the activation of factor C, the single-chain form is cleaved, resulting in an 80 kDa heavy chain and a 43 kDa light chain linked by disulfide bond. The active enzyme is then formed when the light 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Present addresses: Department of Life Science, Faculty of Science, Himeji Institute of Technology, 1479-1 Kanaji, Kamigori, Akou, Hyogo 678-12; "Laboratory of Thrombosis Research, National Cardiovascular Center, 5 Fujishirodai, Suita, Osaka 565. 4 To whom correspondence should be addressed. Abbreviations: Boc, N-tert-butoxycarbonyl; EGF, epidermal growth factor; ELJSA, enzyme linked immunosorbent assay; factor C,
^MC{a^\SS^B^^TX^^S^
chain is subsequently cleaved at a Phe-De bond, producing a 7.9 kDa A chain and a 34 kDa B chain, which are also held together by disulfide bond(s) (6, 7). The presence of a unique Phe-Ile activation site in the light chain indicates that or-chymotrypsin can be used to activate factor C in vitro {7,11). The resulting active factor C (factor C) has the ability to activate factor B and to hydrolyze a synthetic tripeptide substrate Boc-Val-Pro-Arg-pNA. The cDNA sequence of factor C has recently been established in our laboratory. Factor C has a 25-amino-acid leader sequence and the mature protein contains 994 residues, consisting of one Cys-rich domain, one EGF-like domain, one lectin-like domain, five AGP-I-like (Sushi) domains, and a serine protease domain (20). The LPS^ r e ^ o n ^loCflted m ^ ^ . t e r m i n a l 2 5 4 a m i n o ._,-j »,L . • • i_-i_J r n. *fld residues of the heavy chain which 18 composed of the Cy8-nch and EGF domains, and one or two £,GP-I-llke domains (8, 12). In the present study, monoclonal antibodies (mAbs) against factor C have been prepared, in order to elucidate the functional properties of factor C. Moreover, the epibmdin
topesrecogn^byth^antib^ieshavebeenmappedand
50 mM Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl; Tris/HSA buffer, 50 mM Tris-HCl buffer, pH8.0, containing 0.5mg/ml of human serum albumin.
characterized, particularly m reference to the functional domains,
476
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/112/4/476/818333 by university of winnipeg user on 18 January 2019
'Department of Molecular Biology, Graduate School of Medical Science, and "Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812; and '"Panapharm Laboratories Co., Ltd., Uto, Kumamoto 869-04
Monoclonal Antibodies to Limulus Factor C
MATERIALS AND METHODS
Vol. 112, No. 4, 1992
M glycine-HCl, pH 2.8. The concentrations of mAbs were determined by absorbance measurements at 280 nm. The extinction coefficient (jEaJbmn) used was 14.0. Identification of Immunoglobulin Classes—The isotype and subclass of the monoclonal antibodies were determined by ELJSA using factor C-coated plates and the Mouse Immunoglobulin Sets. Preparation of F(ab')% and Fab' Fragments from Monoclonal Antibody 2C12—Seventeen milligrams of mAb 2C12 was digested with pepsin at an enzyme-to-substrate weight ratio of 1 : 100 at 37"C for 8 h, in a total volume of 5.5 ml of 0.1 M sodium citrate buffer, pH 3.5. The sample was concentrated and applied to a Sephadex G-200 column (1x142 cm). The first peak was pooled and dialyzed overnight against 20 mM Tris-HCl buffer, pH8.0. The resulting F(ab')2 fragment was partially reduced by the addition of 2-mercaptoethanol (final cone. 4.7 mM) at 37'C for 75 min under N2 gas. The S-amidomethylation was performed by the addition of iodoacetamide (final cone. 11 mM) on ice for 1 h under N2 gas. Excess iodoacetamide was removed by dialysis against 20 mM Tris-HCl buffer, pH 8.0. Lysyl Endopeptidase Digestion of Factor C—To prepare the LPS-binding fragment, the zymogen factor C was digested with lysyl endopeptidase with an enzyme-tosubstrate weight ratio of 1 : 20 for 1 h at 37'C in sodium acetate buffer, adjusted to pH 8.0 using 1 M Tris-HCl. Western Blotting—Using 2//g each of samples, SDSPAGE was carried out according to the method of Laemmli (12). After electrophoresis, proteins on the gels were transferred to nitrocellulose membranes by using a semidry electroblot apparatus (Trans-Blot®, Bio-Rad) at 10 V for 30 min in 48 mM Tris-39 mM glycine buffer, pH 9.2, containing 1.3 mM SDS and 20% (v/v) methanol. Membranes were then blocked with 20 mM Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl (TBS) and 5% (w/v) nonfat dried milk at room temperature for 1 h. The membranes were washed three times with TBS containing 0.05% Tween 20 and incubated further with mAbs (2^g/ml) diluted with TBS containing 0.2% (w/v) milk at room temperature for 2 h. After washing three times, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-mouse IgG, washed extensively, and subjected to color development using 4-chloro-l-naphthol and 0.1% H2O2 in TBS. Factor C Assay—The amidase activity of factor C generated after activation with LPS was measured using Boc-Val-Pro-Arg-p-nitroanilide (pNA) as a substrate, as described (6). The reaction mixture, comprising 10-20//I of sample in TBS buffer in a total volume of 200 fi\, was preincubated at 37'C for 10 min in the presence or absence of 4//I of LPS (10//g/ml). Then, 50/
Preparation and Properties of Monoclonal Antibodies against Lipopolysaccharide-Sensitive Serine Protease Zymogen, Factor C, from Horseshoe Crab (Tachypleus tridentatus) Hemocytes1 Yoshiki Miura,* Fuminori Tokunaga/' 2 Toshiyuki Miyata,**-3 Mateuko Moriyasu,"* Katsutoshi Yoshikawa,*** and Sadaaki Iwanaga**
Received for publication, April 13, 1992
Seventeen murine monoclonal antibodies (mAbs) against horseshoe crab clotting factor, factor C, were prepared and characterized. When the binding sites of these mAbs were analyzed by immunoblotting, ten mAbs recognized nonreduced factor C, five mAbs were directed against the heavy chain, and two mAbs were directed against the B chain. Three mAbs, 1H4, 2C12, and 2A7, one selected from each group, were used for further study. The mAb 1H4, which recognized only nonreduced factor C molecule, inhibited the factor C activity in a dose-dependent manner. It also inhibited lipopolysaccharide (LPS)- and a-chymotrypsin-mediated activations of the zymogen factor C, suggesting that 1H4 binds close to the active site and/or the substrate-binding site located in the serine protease domain (B chain) of factor C. On the other hand, 2C12 and 2A7 recognized, respectively, an epitope located in the heavy and the B chains, and inhibited LPS-mediated activation of factor C, but not a-chymotrypsin-mediated activation of factor C or factor C activity. Both F(ab')2 and Fab' fragments derived from 2C12 inhibited LPS-mediated activation in the same manner. These three mAbs did not bind with LPS, although a factor C-mAb complex was able to bind LPS, suggesting that the LPS-mediated activation of the zymogen factor C was induced through intermolecular interaction between the LPS-bound factor C molecules. The dissociation constants (Ka) for 1H4, 2C12, and 2A7 binding to factor C were determined as 1.9X10", 0.6x10'°, and 1.8x10'° M, respectively. By using 2C12 and polyclonal antibody against factor C, an enzyme-linked immunosorbent assay for quantitative determination of factor C was established.
Horseshoe crab hemocytes are highly sensitive to Gramnegative bacterial endotoxins, lipopolysaccharides (LPS) (1-3). Exposure of the hemocytes to LPS results in the activation of the intracellular coagulation system, which involves at least three serine protease zymogens named proclotting enzyme (4), factor B (5), and factor C (6-12). The initial activator of the coagulation cascade, factor C, is a zymogen consisting of a single-chain glycoprotein with a molecular weight of 123,000. During the activation of factor C, the single-chain form is cleaved, resulting in an 80 kDa heavy chain and a 43 kDa light chain linked by disulfide bond. The active enzyme is then formed when the light 1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Present addresses: Department of Life Science, Faculty of Science, Himeji Institute of Technology, 1479-1 Kanaji, Kamigori, Akou, Hyogo 678-12; "Laboratory of Thrombosis Research, National Cardiovascular Center, 5 Fujishirodai, Suita, Osaka 565. 4 To whom correspondence should be addressed. Abbreviations: Boc, N-tert-butoxycarbonyl; EGF, epidermal growth factor; ELJSA, enzyme linked immunosorbent assay; factor C,
^MC{a^\SS^B^^TX^^S^
chain is subsequently cleaved at a Phe-De bond, producing a 7.9 kDa A chain and a 34 kDa B chain, which are also held together by disulfide bond(s) (6, 7). The presence of a unique Phe-Ile activation site in the light chain indicates that or-chymotrypsin can be used to activate factor C in vitro {7,11). The resulting active factor C (factor C) has the ability to activate factor B and to hydrolyze a synthetic tripeptide substrate Boc-Val-Pro-Arg-pNA. The cDNA sequence of factor C has recently been established in our laboratory. Factor C has a 25-amino-acid leader sequence and the mature protein contains 994 residues, consisting of one Cys-rich domain, one EGF-like domain, one lectin-like domain, five AGP-I-like (Sushi) domains, and a serine protease domain (20). The LPS^ r e ^ o n ^loCflted m ^ ^ . t e r m i n a l 2 5 4 a m i n o ._,-j »,L . • • i_-i_J r n. *fld residues of the heavy chain which 18 composed of the Cy8-nch and EGF domains, and one or two £,GP-I-llke domains (8, 12). In the present study, monoclonal antibodies (mAbs) against factor C have been prepared, in order to elucidate the functional properties of factor C. Moreover, the epibmdin
topesrecogn^byth^antib^ieshavebeenmappedand
50 mM Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl; Tris/HSA buffer, 50 mM Tris-HCl buffer, pH8.0, containing 0.5mg/ml of human serum albumin.
characterized, particularly m reference to the functional domains,
476
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/112/4/476/818333 by university of winnipeg user on 18 January 2019
'Department of Molecular Biology, Graduate School of Medical Science, and "Department of Biology, Faculty of Science, Kyushu University 33, Higashi-ku, Fukuoka, Fukuoka 812; and '"Panapharm Laboratories Co., Ltd., Uto, Kumamoto 869-04
Monoclonal Antibodies to Limulus Factor C
MATERIALS AND METHODS
Vol. 112, No. 4, 1992
M glycine-HCl, pH 2.8. The concentrations of mAbs were determined by absorbance measurements at 280 nm. The extinction coefficient (jEaJbmn) used was 14.0. Identification of Immunoglobulin Classes—The isotype and subclass of the monoclonal antibodies were determined by ELJSA using factor C-coated plates and the Mouse Immunoglobulin Sets. Preparation of F(ab')% and Fab' Fragments from Monoclonal Antibody 2C12—Seventeen milligrams of mAb 2C12 was digested with pepsin at an enzyme-to-substrate weight ratio of 1 : 100 at 37"C for 8 h, in a total volume of 5.5 ml of 0.1 M sodium citrate buffer, pH 3.5. The sample was concentrated and applied to a Sephadex G-200 column (1x142 cm). The first peak was pooled and dialyzed overnight against 20 mM Tris-HCl buffer, pH8.0. The resulting F(ab')2 fragment was partially reduced by the addition of 2-mercaptoethanol (final cone. 4.7 mM) at 37'C for 75 min under N2 gas. The S-amidomethylation was performed by the addition of iodoacetamide (final cone. 11 mM) on ice for 1 h under N2 gas. Excess iodoacetamide was removed by dialysis against 20 mM Tris-HCl buffer, pH 8.0. Lysyl Endopeptidase Digestion of Factor C—To prepare the LPS-binding fragment, the zymogen factor C was digested with lysyl endopeptidase with an enzyme-tosubstrate weight ratio of 1 : 20 for 1 h at 37'C in sodium acetate buffer, adjusted to pH 8.0 using 1 M Tris-HCl. Western Blotting—Using 2//g each of samples, SDSPAGE was carried out according to the method of Laemmli (12). After electrophoresis, proteins on the gels were transferred to nitrocellulose membranes by using a semidry electroblot apparatus (Trans-Blot®, Bio-Rad) at 10 V for 30 min in 48 mM Tris-39 mM glycine buffer, pH 9.2, containing 1.3 mM SDS and 20% (v/v) methanol. Membranes were then blocked with 20 mM Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl (TBS) and 5% (w/v) nonfat dried milk at room temperature for 1 h. The membranes were washed three times with TBS containing 0.05% Tween 20 and incubated further with mAbs (2^g/ml) diluted with TBS containing 0.2% (w/v) milk at room temperature for 2 h. After washing three times, membranes were incubated at room temperature for 2 h with horseradish peroxidase-conjugated goat anti-mouse IgG, washed extensively, and subjected to color development using 4-chloro-l-naphthol and 0.1% H2O2 in TBS. Factor C Assay—The amidase activity of factor C generated after activation with LPS was measured using Boc-Val-Pro-Arg-p-nitroanilide (pNA) as a substrate, as described (6). The reaction mixture, comprising 10-20//I of sample in TBS buffer in a total volume of 200 fi\, was preincubated at 37'C for 10 min in the presence or absence of 4//I of LPS (10//g/ml). Then, 50/
