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Essential role of mannose-binding lectin-associated serine protease-1 in activation of the complement factor D.

Takahashi M, Ishida Y, Iwaki D, Kanno K, Suzuki T, Endo Y, Homma Y, Fujita T - J. Exp. Med. (2009)

Bottom Line: In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation.Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear.Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan. minolta@fmu.ac.jp

ABSTRACT
The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1- and MASP-3-deficient mouse model (Masp1/3-/-) and found that no activation of the alternative pathway was observed in Masp1/3-/- serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3-/- mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3-/- mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

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Df restores the deficiency of alternative pathway activation in Masp1/3−/− mice. (A) Immunoblot analysis of Bf incubated with the sera of wild-type C57BL/6 (WT), MBL-A, and -C double-deficient (Mbl ) and Masp1/3−/− mice at 37°C for 30 min. Each serum type was incubated with (+) or without (−) 1.25 µg C3(H2O). Serum from Masp1/3−/− mice was also incubated with (+) human Df (0.2 µg). Note: an additional band (∼26 kD) was also detected with Ba, but it was not identified. (B) A hemolytic assay using rabbit erythrocytes was performed. Percent hemolysis induced by sera (25 µl) from wild-type (WT), Masp1/3−/− (M1/3−/−), and Masp1/3−/− mice supplemented with 0.2 µg human Df (huDf) is indicated. (C) A C3 deposition assay on immobilized zymosan was performed. 5 µl of sera from wild-type (WT), Masp2/sMap−/− (M2−/−), and Masp1/3−/− (M1/3−/−) mice supplemented with 0.2 µg human Df (huDf) were analyzed. Data are presented as the means ± SD of three independent experiments.
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fig2: Df restores the deficiency of alternative pathway activation in Masp1/3−/− mice. (A) Immunoblot analysis of Bf incubated with the sera of wild-type C57BL/6 (WT), MBL-A, and -C double-deficient (Mbl ) and Masp1/3−/− mice at 37°C for 30 min. Each serum type was incubated with (+) or without (−) 1.25 µg C3(H2O). Serum from Masp1/3−/− mice was also incubated with (+) human Df (0.2 µg). Note: an additional band (∼26 kD) was also detected with Ba, but it was not identified. (B) A hemolytic assay using rabbit erythrocytes was performed. Percent hemolysis induced by sera (25 µl) from wild-type (WT), Masp1/3−/− (M1/3−/−), and Masp1/3−/− mice supplemented with 0.2 µg human Df (huDf) is indicated. (C) A C3 deposition assay on immobilized zymosan was performed. 5 µl of sera from wild-type (WT), Masp2/sMap−/− (M2−/−), and Masp1/3−/− (M1/3−/−) mice supplemented with 0.2 µg human Df (huDf) were analyzed. Data are presented as the means ± SD of three independent experiments.

Mentions: Circulating C3 is spontaneously converted to a C3b-like product called C3(H2O) upon thioester hydrolysis. In the presence of complement factor B (Bf) and complement factor D (Df), C3(H2O) is able to form the primary C3 convertase C3(H2O)Bb, which triggers the initial step of the alternative pathway (Pangburn and Müller-Eberhard, 1983). To clarify the underlying mechanisms of the aforementioned results, Bf cleavage was investigated by Western blotting. When serum from wild-type C57BL/6 mice was incubated at 37°C for 1 h, a fragment called Ba was cleaved from Bf by limited proteolysis (Fig. 2 A, lane 1). The Ba fragment was also found in serum from Mbl- mice, in which both MBL-A and MBL-C are absent (Fig. 2 A, lane 3; Shi et al., 2004), and exogenous C3(H2O) enhanced Bf activation (Fig. 2 A, lane 2 and 4). No Ba fragment was detected in the following incubation of serum from Masp1/3−/− mice (lane 5), and a faint band of Ba fragment was detected in the presence of exogenous C3(H2O) (Fig. 2 A, lane 6). Because Bf was cleaved by Df, the addition of purified human Df (active form) to Masp1/3−/− mice sera and the following incubation resulted in the cleavage of Bf (Fig. 2 A, lane 7). A very low level of hemolytic activity against rabbit erythrocytes in the sera of Masp1/3−/− mice was compensated by adding active Df (Fig. 2 B), and the low level of C3 deposition on immobilized zymosan using serum from Masp1/3−/− mice was also restored by active Df (Fig. 2 C). These results suggest that Masp1/3−/− mice carry an abnormality in the activation of Df.


Essential role of mannose-binding lectin-associated serine protease-1 in activation of the complement factor D.

Takahashi M, Ishida Y, Iwaki D, Kanno K, Suzuki T, Endo Y, Homma Y, Fujita T - J. Exp. Med. (2009)

Df restores the deficiency of alternative pathway activation in Masp1/3−/− mice. (A) Immunoblot analysis of Bf incubated with the sera of wild-type C57BL/6 (WT), MBL-A, and -C double-deficient (Mbl ) and Masp1/3−/− mice at 37°C for 30 min. Each serum type was incubated with (+) or without (−) 1.25 µg C3(H2O). Serum from Masp1/3−/− mice was also incubated with (+) human Df (0.2 µg). Note: an additional band (∼26 kD) was also detected with Ba, but it was not identified. (B) A hemolytic assay using rabbit erythrocytes was performed. Percent hemolysis induced by sera (25 µl) from wild-type (WT), Masp1/3−/− (M1/3−/−), and Masp1/3−/− mice supplemented with 0.2 µg human Df (huDf) is indicated. (C) A C3 deposition assay on immobilized zymosan was performed. 5 µl of sera from wild-type (WT), Masp2/sMap−/− (M2−/−), and Masp1/3−/− (M1/3−/−) mice supplemented with 0.2 µg human Df (huDf) were analyzed. Data are presented as the means ± SD of three independent experiments.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig2: Df restores the deficiency of alternative pathway activation in Masp1/3−/− mice. (A) Immunoblot analysis of Bf incubated with the sera of wild-type C57BL/6 (WT), MBL-A, and -C double-deficient (Mbl ) and Masp1/3−/− mice at 37°C for 30 min. Each serum type was incubated with (+) or without (−) 1.25 µg C3(H2O). Serum from Masp1/3−/− mice was also incubated with (+) human Df (0.2 µg). Note: an additional band (∼26 kD) was also detected with Ba, but it was not identified. (B) A hemolytic assay using rabbit erythrocytes was performed. Percent hemolysis induced by sera (25 µl) from wild-type (WT), Masp1/3−/− (M1/3−/−), and Masp1/3−/− mice supplemented with 0.2 µg human Df (huDf) is indicated. (C) A C3 deposition assay on immobilized zymosan was performed. 5 µl of sera from wild-type (WT), Masp2/sMap−/− (M2−/−), and Masp1/3−/− (M1/3−/−) mice supplemented with 0.2 µg human Df (huDf) were analyzed. Data are presented as the means ± SD of three independent experiments.
Mentions: Circulating C3 is spontaneously converted to a C3b-like product called C3(H2O) upon thioester hydrolysis. In the presence of complement factor B (Bf) and complement factor D (Df), C3(H2O) is able to form the primary C3 convertase C3(H2O)Bb, which triggers the initial step of the alternative pathway (Pangburn and Müller-Eberhard, 1983). To clarify the underlying mechanisms of the aforementioned results, Bf cleavage was investigated by Western blotting. When serum from wild-type C57BL/6 mice was incubated at 37°C for 1 h, a fragment called Ba was cleaved from Bf by limited proteolysis (Fig. 2 A, lane 1). The Ba fragment was also found in serum from Mbl- mice, in which both MBL-A and MBL-C are absent (Fig. 2 A, lane 3; Shi et al., 2004), and exogenous C3(H2O) enhanced Bf activation (Fig. 2 A, lane 2 and 4). No Ba fragment was detected in the following incubation of serum from Masp1/3−/− mice (lane 5), and a faint band of Ba fragment was detected in the presence of exogenous C3(H2O) (Fig. 2 A, lane 6). Because Bf was cleaved by Df, the addition of purified human Df (active form) to Masp1/3−/− mice sera and the following incubation resulted in the cleavage of Bf (Fig. 2 A, lane 7). A very low level of hemolytic activity against rabbit erythrocytes in the sera of Masp1/3−/− mice was compensated by adding active Df (Fig. 2 B), and the low level of C3 deposition on immobilized zymosan using serum from Masp1/3−/− mice was also restored by active Df (Fig. 2 C). These results suggest that Masp1/3−/− mice carry an abnormality in the activation of Df.

Bottom Line: In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation.Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear.Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan. minolta@fmu.ac.jp

ABSTRACT
The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1- and MASP-3-deficient mouse model (Masp1/3-/-) and found that no activation of the alternative pathway was observed in Masp1/3-/- serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3-/- mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3-/- mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

Show MeSH
Related in: MedlinePlus