Proteolytic Activities of Two Types of Mannose-Binding Lectin-Associated Serine Protease

Mannose (or mannan)-binding lectin (MBL) is an oligomeric serum lectin that plays a role in innate immunity by activating the complement system. In human, two types of MBL-associated serine protease (MASP-1 and MASP-2) and a truncated protein of MASP-2 (small MBL-associated protein; sMAP or MAp19) are complexed with MBL. To clarify the proteolytic activities of MASP-1 and MASP-2 against C4, C2, and C3, we isolated these two types of MASP in activated forms from human serum by sequential affinity chromatography. On an anti-MASP-1 column, MASP-2 passed through the column in the presence of EDTA and high salt concentration, whereas MASP-1 was retained. Isolated MASP-1 and MASP-2 exhibited proteolytic activities against C3 and C4, respectively. C2 was activated by both MASPs. C1 inhibitor (C1 INH), an inhibitor for C1r and C1s, formed equimolar complexes with MASP-1 and MASP-2 and inhibited their proteolytic activities.

M annose (or mannan)-binding lectin (MBL), 3 a member of the collectins (1), is a Ca 2ϩ -dependent serum lectin that recognizes carbohydrates such as mannose and N-acetylglucosamine (GlcNAc) (2) on the surfaces of pathogens and plays a role in innate immunity through activating complement (3)(4)(5). MBL is an oligomer of subunits composed of three identical polypeptide chains comprising a cysteine-rich, a collagen-like, a neck, and a carbohydrate recognition domain. MBL forms a complex with C1r/C1s-like serine proteases termed MASPs (MBLassociated serine proteases), and activates complement via the lectin pathway (6,7). To date, two types of MASP, MASP-1 (8,9) and MASP-2 (10,11), have been identified in human MBL preparations. MASP-1, MASP-2, C1r, and C1s constitute a subfamily of the serine protease family (12). Their structure shares six domains, a first CUB domain, an epidermal growth factor-like domain, a second CUB domain, two complement control protein domains, and a serine protease domain (13,14). Recently, a protein designated sMAP (small MBL-associated protein) (15) or MAp19 (MBL-associated plasma protein of 19 kDa) (16) has been identified. sMAP is identical with MASP-2 through the first two domains followed by four unique amino acids at the C-terminal, which are derived from a specific exon of the MASP-2 gene. The physiological role of sMAP remains unknown. In blood, MBL forms complexes (MBL-complexes) with the proenzyme forms of MASP-1 and MASP-2 and with sMAP. Upon binding of MBL to its ligands, MASPs are activated by a split in the polypeptide chain, generating two polypeptides linked by a disulfide bond, thus acquiring proteolytic activities. It has recently been demonstrated that a serum GlcNAc-binding lectin termed ficolin/P35 with collagen-like and fibrinogen-like domains is also associated with MASPs and sMAP (17).
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.

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 CaCl 2 , 0.2 mM NPGB, 20 M 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 CaCl 2 , 0.2 mM NPGB, and 20 M 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 l of sample containing MASP-1 or MASP-2 diluted in MGVB was incubated with 50 l of C4 (two site-forming units, SFU) at 37°C for 30 min. The reaction mixtures were further incubated for 60 min with 100 l of 50fold-diluted C4-deficient guinea pig serum and 100 l of sheep erythrocytes (10 8 /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 ϭ Ϫln(1 Ϫ y) was calculated. The percentage consumption was determined by the following formula: % consumption ϭ (Z1 Ϫ Z2)/Z1 ϫ 100, where Z1 ϭ z value in the absence of sample and Z2 ϭ z value in the presence of sample.
EA bearing human C4b (EAC4b) was prepared as described previously (23). Fifty microliters of samples, 50 l of oxidized human C2 (2 SFU), and 100 l of EAC4b (10 8 /ml) were incubated in MGVB at 30°C for 10 min, and then 200 l 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 l of samples and 10 l of human C3 (2 g) 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 l of various amounts of C1 INH diluted in MGVB at 37°C for 15 min and then with 50 l 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:

Separation of MASP-1 and MASP-2
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 ϳ93 kDa and 70 kDa, respectively, indicating that both MASPs were in proenzyme forms.
Based on the results that proenzymes MASP-1 and MASP-2 were separated on the anti-MASP-1 column, we next isolated MASP-1 and MASP-2 in activated forms. The MBL-complex was prepared utilizing mannan and anti-MBL columns. After dialysis against a buffer containing EDTA and 1 M NaCl, the MBL-complex from the anti-MBL column was applied to the anti-MBL column again. In this buffer, MASP-1, MASP-2, and sMAP passed through the column and thus separated from MBL. This preparation was finally fractionated on an anti-MASP-1 column. MASP-2 passed through the column, whereas MASP-1 was retained on the column and could be eluted with an acidic buffer as was the case with the MASP proenzymes (Fig. 2). 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.

Proteolytic activities of MASP-1 and MASP-2
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.

Complex formation between MASPs and C1 INH
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

Inhibition of MASP function by C1 INH
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).   activities of MASP-2 were inhibited by C1 INH in a dose-dependent manner.

Discussion
The binding between C1q, C1r, and C1s in the C1 complex is facilitated by Ca 2ϩ , and the complex is formed in a 1:2:2 stoichiometry. On the other hand, the mode of binding and stoichiometry of the complex composed of MBL, MASP-1, MASP-2, and sMAP remains unsolved. In the present report, we showed that MASP-1, MASP-2, and sMAP dissociate from MBL in the presence of EDTA and high concentration of salt (1 M NaCl). Several lines of evidence have revealed that EDTA alone is insufficient, and both EDTA and high concentration of salt are required for the dissociation of MBL from the other components, suggesting that the complex formation is facilitated by a combination of Ca 2ϩ and presumably electrostatic interactions (28,29). When the mixture of MASP-1, MASP-2, and sMAP from the anti-MBL column was applied to an anti-MASP-1 column with buffer containing EDTA and 1 M NaCl, MASP-2 was recovered in the pass-through fractions, whereas MASP-1 was retained on the column and could be eluted with an acidic buffer. Two explanations can be proposed for the separation of MASP-1 and MASP-2 on this column; MASP-1 and MASP-2 form a complex in a Ca 2ϩ -dependent manner or, alternatively, MASP-1 and MASP-2 are independently complexed with MBL.
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 (␣ 2 M) that is a protease inhibitor in blood (34). The effect of ␣ 2 M 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 ␣ 2 M 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.