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*
Department of Biochemistry, Fukushima Medical University School of Medicine, Fukushima, Japan;
Department of Medical Microbiology and Immunology, The Bartholin Building, University of Aarhus, Aarhus, Denmark;
Division of Clinical Pathology, Hokkaido Institute of Public Health, Sapporo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MASP-1 was first discovered and characterized as a serine protease capable of activating C4, C2 (8), and C3 (18). In a subsequent report, Thiel et al. (10) separated human MASP-1 and MASP-2 by SDS-PAGE and demonstrated that when blotted onto a membrane MASP-2 activates C4, whereas MASP-1 does not.
C1 inhibitor (C1 INH) is a plasma protein that belongs to the serpin superfamily of serine protease inhibitors, and is in blood involved in the regulation of proteolytic systems such as the coagulation, fibrinolytic, and complement systems (19). In the complement system, C1 INH exhibits inhibitory activities against C1r and C1s, thus playing a role in the regulation of classical pathway activation. The importance of C1 INH is revealed by genetically determined C1 INH deficiency that causes hereditary angioedema (HAE).
The present study describes the separation of MASP-1 and MASP-2 and their activities against complement components. The effects of C1 INH on the proteolytic activities of MASP-1 and MASP-2 were also examined to elucidate the regulation mechanism of the lectin pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mannan from Saccharomyces cerevisiae was purchased from Sigma (St. Louis, MO). (Amidinophenyl)methanesulfonyl fluoride (p-APMSF) and mannose were from Wako Pure Chemical Industries (Osaka, Japan). p-Nitrophenyl-p-guanidinobenzoate (NPGB) was from Merck (Rahway, NJ). CNBr-activated Sepharose 4B was from Amersham Pharmacia (Uppsala, Sweden). Mouse mAbs against MBL (3E7) (20) and MASP-1 (1E2) (21) and rabbit polyclonal Abs against a synthetic peptide representing 19 C-terminal amino acids of MASP-1 (10) were prepared as described previously. Anti-MASP-2 Ab was from rabbits immunized with a synthetic peptide corresponding to the first 20 N-terminal amino acid residues of MASP-2 that was conjugated to a multiple Ag peptide backbone (22). Coupling of mannan, anti-MBL (3E7), or anti-MASP-1 (1E2) to CNBr-activated Sepharose 4B was performed according to the manufacturer’s instructions. Human C3, C4 (23), C2 (24), oxidized C2 (25), and C1 INH (26) were prepared as previously described. Veronal-buffered saline (VB) is a 10-mM solution of Veronal containing 0.148 M NaCl (pH 7.4). EDTA-GVB is VB supplemented with 10 mM EDTA and 0.1% gelatin. MGVB is a 5-mM solution of Veronal containing 0.074 M NaCl, 0.1% gelatin, 2.3% mannitol, 2 mM CaCl2, and 0.5 mM MgCl2.
Preparation of human MASP-1 and MASP-2
MASP-1 and MASP-2 in proenzyme forms were isolated from human serum as described previously (9, 15, 27). In brief, human serum was passed through a yeast mannan-Sepharose column using a 10 mM imidazole buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl2, 0.2 mM NPGB, 20 µM p-APMSF, and 2% mannitol. Proenzymes MASP-1 and MASP-2 complexed with MBL were eluted with the above buffer containing 0.3 M mannose. To separate proenzymes MASP-1 and MASP-2 from MBL, preparations containing the complex were applied to anti-MBL-Sepharose and then MASPs were eluted with imidazole buffer containing 20 mM EDTA and 1 M NaCl. Finally, proenzymes MASP-1 and MASP-2 were separated by passing through anti-MASP-1-Sepharose in the same buffer as used for the anti-MBL-Sepharose. MASP-2 was recovered in the effluents, whereas MASP-1 was eluted with 0.1 M glycine buffer (pH 2.2).
Human MBL-complexes, in which MASP-1 and MASP-2 were in activated forms, were isolated from serum. For this, human serum was first applied to a mannan-Sepharose column equilibrated with 50 mM Tris buffer (pH 6.0) containing 0.2 M NaCl, 20 mM CaCl2, 0.2 mM NPGB, and 20 µM p-APMSF. After washing with starting buffer without NPGB and p-APMSF, elution was conducted with the same buffer containing 0.3 M mannose. The MBL-complex eluate was next applied to the anti-MBL-Sepharose column equilibrated with the same buffer. MBL-complexes were eluted from the column with glycine buffer. After dialysis against 50 mM Tris buffer containing 1 M NaCl, 20 mM EDTA, the MBL-complex preparation was applied to an anti-MBL-Sepharose column. The effluent contained a mixture of MASP-1, MASP-2, and sMAP, whereas MBL was retained and subsequently eluted with glycine buffer. The preparation containing MASP-1, MASP-2, and sMAP was applied to an anti-MASP-1-Sepharose column equilibrated with the same buffer as used for the second anti-MBL-Sepharose. At this step, MASP-2 passed through, whereas MASP-1 was retained on the column and eluted with glycine buffer. sMAP was found in both the MASP-1 and the MASP-2 fractions. The fractions containing MASP-1 or MASP-2 were pooled and used to study the effects of C1 INH on MASP activity.
SDS-PAGE and immunoblotting
SDS-PAGE was performed according to the Laemmli method. After transferring proteins from the gels to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blots were probed with anti-MASP-1 peptide or anti-MASP-2 peptide Abs. Peroxidase-conjugated anti-rabbit IgG was used as a second Ab, and the blot was developed with a Konica Immunostain kit (Konica, Tokyo, Japan).
Proteolytic activities of MASP-1 and MASP-2
C4 consumption was assayed as described previously (8). In brief, 50 µl of sample containing MASP-1 or MASP-2 diluted in MGVB was incubated with 50 µl of C4 (two site-forming units, SFU) at 37°C for 30 min. The reaction mixtures were further incubated for 60 min with 100 µl of 50-fold-diluted C4-deficient guinea pig serum and 100 µl of sheep erythrocytes (108/ml) bearing anti-sheep erythrocytes Abs (EA). The lytic reaction was terminated by the addition of 1 ml of EDTA-GVB. After centrifugation, the OD of the supernatant was determined at 414 nm. The hemolytic rate (y) and the average number of hemolytic sites per cell (z) defined as z = -ln(1 - y) was calculated. The percentage consumption was determined by the following formula: % consumption = (Z1 - Z2)/Z1 x 100, where Z1 = z value in the absence of sample and Z2 = z value in the presence of sample.
EA bearing human C4b (EAC4b) was prepared as described previously (23). Fifty microliters of samples, 50 µl of oxidized human C2 (2 SFU), and 100 µl of EAC4b (108/ml) were incubated in MGVB at 30°C for 10 min, and then 200 µl of 50-fold-diluted guinea pig serum with EDTA-GVB (C-EDTA) was added to the reaction mixture as a source of C3 to C9. After additional incubation at 37°C for 60 min, 1 ml of EDTA-GVB was added to terminate the reaction. From the OD determined at 414 nm, z was calculated as described above.
C3 activation was assayed as described previously (18). In brief, 10 µl of samples and 10 µl of human C3 (2 µg) in VB was incubated at 37°C for 60 min, and the reaction mixture was subjected to SDS-PAGE (7.5% gel) under reducing conditions.
Complex formation between MASPs and C1 INH
MASP-1 or MASP-2 was incubated with C1 INH at 37°C for 30 min. The mixtures were then subjected to SDS-PAGE under nonreducing conditions followed by immunoblotting.
Effect of C1 INH on the proteolytic activities of MASPs
Five microliters of fractions containing MASP-2 in MGVB were incubated with 45 µl of various amounts of C1 INH diluted in MGVB at 37°C for 15 min and then with 50 µl of C4 at 37°C for 30 min. Residual hemolytic activity of C4 was assayed as described above (C4 consumption), and the effect of C1 INH on MASP-2-mediated C4 activation was expressed as the percentage inhibition by the following formula: % inhibition = (Z3 - Z2)/(Z1 -Z2) x 100, where Z1 = z value in the absence of MASP-2 and C1 INH, Z2 = z value in the presence of MASP, and Z3 = z value in the presence of MASP and C1INH. For inhibition of C2 activation, 25 µl of MASP-1 or MASP-2 were incubated with 25 µl of various amounts of C1 INH, 50 µl of oxidized C2, and 100 µl of EAC4b at 30°C for 10 min and then with C-EDTA at 37°C for 1 h, and the effect of C1 INH on MASP-1 and on MASP-2 was expressed as the percentage inhibition by the following formula: % inhibition = Z2/(Z1 - Z2) x 100, where Z1 = z value in the absence of MASP-2 and Z2 = z value in the presence of MASP-2.
For direct observation of the effect of C1 INH on MASP-1, 10 µl of MASP-1 were incubated with 10 µl of various amounts of C1 INH at 37°C for 30 min and then with 10 µl of C3 (2 µg) for 60 min. The reaction mixtures were analyzed by SDS-PAGE (7.5% gel) under reducing conditions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To obtain human MASP-1 and MASP-2 in proenzyme forms, the
MBL-complex was first prepared from serum using a mannan column in the
presence of serine protease inhibitors. This preparation also contained
IgG and IgM. Further purification was achieved using an anti-MBL
column. The MBL-complex was bound to the anti-MBL column, and MASPs
and sMAP were then eluted with EDTA at a high salt concentration,
whereas MBL was retained on the column. Finally, the eluate containing
MASPs and sMAP was subjected to affinity chromatography on an
anti-MASP-1 column. At this step, MASP-2 passed through the column,
whereas MASP-1 was retained on the column and could subsequently be
eluted with an acidic buffer (Fig. 1
).
Most of sMAP coeluted with MASP-1. MASP-1 and MASP-2 obtained in this
way showed a single band under reducing conditions with molecular size
of 
93 kDa and 70 kDa, respectively, indicating that both MASPs were
in proenzyme forms.
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). Both MASP-1
and MASP-2 were in activated forms as revealed by the presence of the L
chain of MASP-1 and the H chain of MASP-2. sMAP was found to copurify
with both MASP-1 and MASP-2.
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Preparations containing either MASP-1 or MASP-2 in activated forms
were tested for proteolytic activities against C4, C2, and C3. C4
consumption and C2 activation by MASPs were determined hemolytically as
described in Materials and Methods. C3 cleavage by MASP was
directly assessed by SDS-PAGE. As shown in Fig. 3, C4 consumption was observed with the
fractions containing MASP-2 but not with those containing MASP-1. Both
MASP-1 and MASP-2 activated C2. C3 cleavage with an appearance of the
-chain was noted for MASP-1 but not for MASP-2. In contrast with the
activated forms of MASPs, proenzymes MASP-1 and MASP-2 showed no
proteolytic activities against C4, C2, and C3 (data not shown),
indicating that at the conditions of the experiment no activation of
the proenzymes occurred.
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C1 INH forms stable complexes with C1s and C1r in a 1:1 ratio and
inhibits their proteolytic activities. To determine the effect of C1
INH on MASP-1 or MASP-2, we first tested for covalent complex formation
between C1 INH and MASPs in activated forms. C1 INH was incubated with
MASP-1 or MASP-2 in activated forms at 37°C for 1 h and then
subjected to SDS-PAGE followed by immunoblotting. As shown in Fig. 4A, a novel band with an
apparent m.w. of 196 kDa reacting with anti-MASP-1 Ab appeared
after incubation of C1 INH with MASP-1. The molecular size of this band
almost matched the sum of MASP-1 (81 kDa) and C1 INH (98 kDa).
Similarly, incubation of MASP-2 (63 kDa) with C1 INH resulted in an
appearance of a new band (175 kDa) (Fig. 4B). These results
indicate that C1 INH formed equimolar complexes with MASP-1 and
MASP-2.
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We next determined whether C1 INH inhibits the proteolytic
activities of MASP-1 and MASP-2 in activated forms. The proteolytic
activities of MASP-1 against C3 and C2 were examined in the presence of
various amounts of C1 INH. As shown in Fig. 5A, C3 cleavage by MASP-1 was
inhibited by C1 INH in a dose-dependent manner. Similarly, C1 INH
inhibited C2 activation mediated by MASP-1 (Fig. 5B). Fig. 5, C and D, depict the results of the effects of
C1 INH on MASP-2 activities against C4 and C2. Both activities of
MASP-2 were inhibited by C1 INH in a dose-dependent manner.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Figs. 1
and 2, sMAP copurified on the anti-MASP-1
column with both MASP-1 and MASP-2 when they were in activated forms,
whereas most of the sMAP coeluted with MASP-1 when the MASPs were in
proenzyme forms. The reason for this difference in the behavior of sMAP
remains unclear. One possibility is that a long exposure of MASP-1-sMAP
to EDTA and 1 M NaCl during dialysis in the purification step of
activated MASPs allowed some dissociation of MASP-1 and sMAP.
Alternatively, it could be suggested that sMAP has a lower affinity for
activated MASP-1 than for unactivated MASP-1.
Isolated MASP-1 and MASP-2 in activated forms exhibited proteolytic activities against C3 and C4, respectively. The specificity of MASP-2 for C4 is consistent with a previous report (10). Both MASPs activated C2. In this respect, the function of MASP-2 resembles C1s in the C1 complex, whereas MASP-1 shows unique proteolytic activities. Analysis of the cDNA of MASP and C1r/C1s serine protease domains from human, mouse, hamster, Xenopus, carp, shark, lamprey, and ascidian revealed that the MASP/C1r/C1s family falls into two groups (30, 31). The first group, termed "TCN type," where the serine residue in the active center is encoded by TCN, encompasses human MASP-1, mouse MASP-1, Xenopus MASP-1, ascidian MASPa, and MASPb. The second group, termed "AGY type," where the serine residue in the active center is encoded by AGY, encompasses human MASP-2, human C1r/C1s, hamster C1s, mouse MASP-2, Xenopus MASP-2, carp MASP, shark MASP, and lamprey MASP. The TCN type possesses a so called "histidine loop" structure, whereas the AGY type does not. It is speculated that the AGY type diverged from the TCN type in the evolution of the MASP/C1r/C1s family (30, 32). The ascidians appear to lack the classical pathway C4 and C2, and the function of ascidian MASPs may thus be restricted to cleavage of C3 (33). This type of substrate selectivity has been preserved in human MASP-1, which, like ascidian MASPs, possesses the histidine loop. The C4 cleaving activity of human MASP-2 and C1s could be speculated to be related to the cutout of the histidine loop. The split exon nature of ascidian MASPs and MASP-1 (30), and the TCN codon contrasting to MASP-2 and C1r/C1s are features of no structural consequence to the proteins, but most useful when trying to sort out the phylogeny. Although the stoichiometry of the MBL-complex and activation mechanism remain unsolved, MASP-2 in the complex is likely to possess the same function as C1s, which cleaves C4 and C2 resulting in the formation of C3 convertase, C4b2a. On the other hand, MASP-1 directly cleaves C3 into C3a and C3b, the latter of which initiates the alternative complement pathway (18) and also acts as an opsonin. The physiological significance of the observed C2 activating-capacity of MASP-1 is unclear. It is also unknown which MASP is more active in cleaving C2 on a molar basis, since the presence of sMAP in both MASP-1 and MASP-2 preparations does not allow quantitative analysis of MASPs. However, it should be noted that more C1 INH was required for preventing proteolysis of C2 by MASP-1 than by MASP-2, suggesting that the proteolytic activity of MASP-1 against C2 might be lower than that of MASP-2.
Unlike C1r or C1s, MASP-1 forms a complex with
2-macroglobulin (2M) that is a protease
inhibitor in blood (34). The effect of 2M
on MASP-2 is to be elucidated. In this report, we demonstrated that C1
INH inhibited the proteolytic activities of both MASP-1 and MASP-2 by
forming stable complexes in a 1:1 stoichiometry as do C1r and C1s,
indicating a function in regulation of lectin pathway activation. Wong
et al. (35) also observed that a mixture of activated
MASP-1 and MASP-2 interacts with C1 INH, resulting in the formation of
complexes between C1 INH and each MASP. In the C1 complex in blood, C1
INH associates noncovalently with proenzyme C1r to prevent its
autoactivation. Upon binding of C1 to immune complexes, C1 INH
dissociates from proenzyme C1r, resulting in the autoactivation of C1r
and the subsequent activation of C1s. Thus, C1 INH also modulates C1
activation by inhibiting the autoactivation of C1r. If MASP-1 and/or
MASP-2 autoactivate, it is possible that their autoactivation is also
regulated by C1 INH in a manner similar to C1r. The present and
previous studies (34, 36) suggest that C1 INH regulates
both the classical and lectin pathways and 2M regulates
the latter.
The MBL-complex and the C1 complex appear to be similar in that the serine proteases involved in each complex have specific proteolytic activities. However, several features are different between the MBL-complex and C1. First, the MBL-complex possesses sMAP, which has no equivalent in C1, although its role in the complex remains unsolved. Second, as discussed above, it is possible that MASP-1 and MASP-2 are independently associated with MBL.
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Acknowledgments |
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Footnotes |
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2 Address correspondence to Dr. Misao Matsushita, Department of Biochemistry, Fukushima Medical University School of Medicine, 1-Hikariga-oka, Fukushima 960-1295, Japan.
3 Abbreviations used in this paper: MBL, mannose (or mannan)-binding lectin; GVB, gelatin-containing Veronal-buffered saline; VB, Veronal-buffered saline; EDTA-GVB, gelatin-Veronal buffer containing EDTA; C-EDTA, guinea pig serum diluted with EDTA-GVB; C1 INH, C1 inhibitor; CUB, C1r/C1s/Uegf/bone morphogenetic protein 1; EA, sheep erythrocytes sensitized with Ab; EAC4b, EA bearing guinea pig C1 and human C4b; sMAP, small MBL-associated protein; MAp19, MBL-associated plasma protein of 19 kDa; MASP, MBL-associated serine protease; MBL-complex, a complex consisting of MBL, MASP-1, MASP-2, and sMAP; MGVB, gelatin-Veronal buffer containing mannitol, CaCl2, and MgCl2; NPGB, p-nitrophenyl-p-guanidinobenzoate; p-APMSF, (amidinophenyl)methanesulfonyl fluoride.
Received for publication April 10, 2000. Accepted for publication June 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin binds to and inhibits mannose-binding protein-associated serine protease. Int. Immunol. 7:1579.This article has been cited by other articles:
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|
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|
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|
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G. Ambrus, P. Gal, M. Kojima, K. Szilagyi, J. Balczer, J. Antal, L. Graf, A. Laich, B. E. Moffatt, W. Schwaeble, et al. Natural Substrates and Inhibitors of Mannan-Binding Lectin-Associated Serine Protease-1 and -2: A Study on Recombinant Catalytic Fragments J. Immunol., February 1, 2003; 170(3): 1374 - 1382. [Abstract] [Full Text] [PDF]
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M. Kuraya, M. Matsushita, Y. Endo, S. Thiel, and T. Fujita Expression of H-ficolin/Hakata antigen, mannose-binding lectin-associated serine protease (MASP)-1 and MASP-3 by human glioma cell line T98G Int. Immunol., January 1, 2003; 15(1): 109 - 117. [Abstract] [Full Text] [PDF]
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S. Cseh, L. Vera, M. Matsushita, T. Fujita, G. J. Arlaud, and N. M. Thielens Characterization of the Interaction Between L-Ficolin/P35 and Mannan-Binding Lectin-Associated Serine Proteases-1 and -2 J. Immunol., November 15, 2002; 169(10): 5735 - 5743. [Abstract] [Full Text] [PDF]
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G. Vinci, N. J. Lynch, C. Duponchel, T.-M. Lebastard, G. Milon, C. Stover, W. Schwaeble, and M. Tosi In Vivo Biosynthesis of Endogenous and of Human C1 Inhibitor in Transgenic Mice: Tissue Distribution and Colocalization of Their Expression J. Immunol., November 15, 2002; 169(10): 5948 - 5954. [Abstract] [Full Text] [PDF]
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L. Zhao, Y. Ohtaki, K. Yamaguchi, M. Matsushita, T. Fujita, T. Yokochi, H. Takada, and Y. Endo LPS-induced platelet response and rapid shock in mice: contribution of O-antigen region of LPS and involvement of the lectin pathway of the complement system Blood, October 16, 2002; 100(9): 3233 - 3239. [Abstract] [Full Text] [PDF]
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Y. Endo, M. Takahashi, M. Kuraya, M. Matsushita, C. M. Stover, W. J. Schwaeble, and T. Fujita Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter Int. Immunol., October 1, 2002; 14(10): 1193 - 1201. [Abstract] [Full Text] [PDF]
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M. Matsushita, M. Kuraya, N. Hamasaki, M. Tsujimura, H. Shiraki, and T. Fujita Activation of the Lectin Complement Pathway by H-Ficolin (Hakata Antigen) J. Immunol., April 1, 2002; 168(7): 3502 - 3506. [Abstract] [Full Text] [PDF]
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A. Roos, A. J. Nauta, D. Broers, M. C. Faber-Krol, L. A. Trouw, J. W. Drijfhout, and M. R. Daha Specific Inhibition of the Classical Complement Pathway by C1q-Binding Peptides J. Immunol., December 15, 2001; 167(12): 7052 - 7059. [Abstract] [Full Text] [PDF]
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 H. Jiang, E. Wagner, H. Zhang, and M. M. Frank Complement 1 Inhibitor Is a Regulator of the Alternative Complement Pathway J. Exp. Med., December 3, 2001; 194(11): 1609 - 1616. [Abstract] [Full Text] [PDF]
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Y. Endo, T. Yokochi, M. Matsushita, T. Fujita, and H. Takada Complement-dependent platelet degradation and anaphylactoid shock in mice induced by lipopolysaccharide carrying the mannose homopolymer Innate Immunity, December 1, 2001; 7(6): 451 - 455. [Abstract] [PDF]
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 I. Celik, C. Stover, M. Botto, S. Thiel, S. Tzima, D. Kunkel, M. Walport, W. Lorenz, and W. Schwaeble Role of the Classical Pathway of Complement Activation in Experimentally Induced Polymicrobial Peritonitis Infect. Immun., December 1, 2001; 69(12): 7304 - 7309. [Abstract] [Full Text] [PDF]
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H. Sekine, A. Kenjo, K. Azumi, G. Ohi, M. Takahashi, R. Kasukawa, N. Ichikawa, M. Nakata, T. Mizuochi, M. Matsushita, et al. An Ancient Lectin-Dependent Complement System in an Ascidian: Novel Lectin Isolated from the Plasma of the Solitary Ascidian, Halocynthia roretzi J. Immunol., October 15, 2001; 167(8): 4504 - 4510. [Abstract] [Full Text] [PDF]
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 J. E. Jordan, M. C. Montalto, and G. L. Stahl Inhibition of Mannose-Binding Lectin Reduces Postischemic Myocardial Reperfusion Injury Circulation, September 18, 2001; 104(12): 1413 - 1418. [Abstract] [Full Text] [PDF]
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A. Roos, L. H. Bouwman, D. J. van Gijlswijk-Janssen, M. C. Faber-Krol, G. L. Stahl, and M. R. Daha Human IgA Activates the Complement System Via the Mannan-Binding Lectin Pathway J. Immunol., September 1, 2001; 167(5): 2861 - 2868. [Abstract] [Full Text] [PDF]
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N. M. Thielens, S. Cseh, S. Thiel, T. Vorup-Jensen, V. Rossi, J. C. Jensenius, and G. J. Arlaud Interaction Properties of Human Mannan-Binding Lectin (MBL)-Associated Serine Proteases-1 and -2, MBL-Associated Protein 19, and MBL J. Immunol., April 15, 2001; 166(8): 5068 - 5077. [Abstract] [Full Text] [PDF]
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 M. J. Walport Complement- First of Two Parts N. Engl. J. Med., April 5, 2001; 344(14): 1058 - 1066. [Full Text] [PDF]
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A. Kenjo, M. Takahashi, M. Matsushita, Y. Endo, M. Nakata, T. Mizuochi, and T. Fujita Cloning and Characterization of Novel Ficolins from the Solitary Ascidian, Halocynthia roretzi J. Biol. Chem., June 1, 2001; 276(23): 19959 - 19965. [Abstract] [Full Text] [PDF]
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V. Rossi, S. Cseh, I. Bally, N. M. Thielens, J. C. Jensenius, and G. J. Arlaud Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2 J. Biol. Chem., October 26, 2001; 276(44): 40880 - 40887. [Abstract] [Full Text] [PDF]
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), C4 consumption. Samples of each
fraction were incubated with C4 and residual hemolytic activities of C4
were determined. Middle (
), C2 activation. Samples of
each fraction were incubated with EAC4b and C2 and then with C3 to C9
(guinea pig serum diluted with EDTA-GVB). From hemolytic rates,
z was determined as described in Materials and
Methods. Bottom (SDS-PAGE), C3 cleavage. Samples
of each fraction or buffer (left lane) were incubated with C3 and the
reaction mixtures were subjected to SDS-PAGE (7.5% gel) under reducing
conditions. The gel was stained with Coomassie Brilliant Blue
R-250.
