Substrate Specificities of Recombinant Mannan-binding Lectin-associated Serine Proteases-1 and -2

Abstract

Mannan-binding lectin (MBL)-associated serine proteases-1 and 2 (MASP-1 and MASP-2) are homologous modular proteases that each interact with MBL, an oligomeric serum lectin involved in innate immunity. To precisely determine their substrate specificity, human MASP-1 and MASP-2, and fragments from their catalytic regions were expressed using a baculovirus/insect cells system. Recombinant MASP-2 displayed a rather wide, C1s-like esterolytic activity, and specifically cleaved complement proteins C2 and C4, with relative efficiencies 3-and 23-fold higher, respectively, than human C1s. MASP-2 also showed very weak C3 cleaving activity. Recombinant MASP-1 had a lower and more restricted esterolytic activity. It showed marginal activity toward C2 and C3, and no activity on C4. The enzymic activity of both MASP-1 and MASP-2 was specifically titrated by C1 inhibitor, and abolished at a 1:1 C1 inhibitor:protease ratio. Taken together with previous findings, these and other data strongly support the hypothesis that MASP-2 is the protease that, in association with MBL, triggers complement activation via the MBL pathway, through combined self-activation and proteolytic properties devoted to C1r and C1s in the C1 complex. In view of the very low activity of MASP-1 on C3 and C2, our data raise questions about the implication of this protease in complement activation.Mannan-binding lectin (MBL) 1 is an oligomeric C-type lectin that recognizes arrays of neutral carbohydrates such as mannose and N-acetylglucosamine on the surface of pathogenic microorganisms (2). This selectivity endows MBL with the ability to discriminate self from infectious non-self, and confers this "ante-antibody" a major role in innate immunity, as underlined by numerous clinical reports indicating that MBL deficiency is linked with increased susceptibility to infectious diseases (3-5). In addition to its role as an opsonin (3), MBL has devised the ability to associate to several modular proteases termed MASPs (MBL-associated serine proteases) (6 -9). A single MASP entity was initially identified, and characterized as a protease with the ability to cleave complement proteins C4, C2, and C3 (7, 10). Further studies by Thiel et al. (6) revealed that MASP was indeed a mixture of two related but distinct proteases, MASP-1 and MASP-2, and that only the latter had the ability to cleave C4. A third protein component MAp19, arising from alternative splicing of the MASP-2 gene (11, 12), and very recently a further protease MASP-3 (13) were also shown to be associated with MBL.MASP-1 and MASP-2 show a domain organization identical to that of C1r and C1s, the enzymatic components of the C1 complex of complement (14), with an N-terminal CUB module (15) followed by an epidermal growth factor-like module, a second CUB module, two contiguous CCP modules (16), and a C-terminal chymotrypsin-like serine protease domain (see Fig. 1). By analogy with human C1s, it may be anticipated that the proteolytic activity and specificity of the MASPs is defined by the two CCP modules ...

Activation of Complement Component C5

“…Based on the crystal structure of the CUB1-EGF-CUB2 region of human MASP-2, Feinberg et al proposed a model in which a MASP-2 dimer was shown to easily dock inside a MBL dimer with the serine protease domain of MASP-2 near the flexible ␣-helical neck joint (15). Kinetic studies have shown that although C1s and MASP-2 have comparable C2-cleaving activities, MASP-2 is more efficient than C1s in cleaving C4 due to a 26-fold lower K m for the substrate (K m ϭ 0.074 M and 1.92 M, respectively) (18). Thus even though the stoichiometry of the MBL⅐MASP-2 complex of the lectin pathway might be similar to that reported for the C1 complex of the classical pathway in which one C1q hexamer binds to one C1s dimer (34,59), the reports suggest that the lectin pathway is more efficient than the classical pathway in cleaving C4.…”

“…Cleavage of C4 and C2 are mediated by C1s in the C1r 2 -C1s 2 tetramer, which gets activated upon the binding of C1 to antigen-antibody complexes (33,34). Similarly, cleavage of C4 and C2 upon activation of the lectin pathway is mediated by MASP-2 in the M1 complex (13,(17)(18)(19)35). Each type of MASP has been reported to form a homodimer (10,36,37), but the stoichiometry of MASPs in association with MBL has yet to be established.…”

“…Each type of MASP has been reported to form a homodimer (10,36,37), but the stoichiometry of MASPs in association with MBL has yet to be established. Crystallography studies have shown that, even though C1s and MASP-2 have identical substrate specificity, different enzyme-substrate interactions sites are recognized by C1s and MASP-2 for C4 and C2 (38), and kinetic studies have shown that, although C1s and MASP-2 have comparable C2 cleaving activities, MASP-2 is more efficient than C1s in cleaving C4 due to a 26-fold lower K m for the substrate (18).…”

“…Binding of MBL⅐MASP complexes to sugar residues present on the cell surfaces of microorganisms results in a conformational change of MBL (15,16), which activates MASP-2. Activated MASP-2 cleaves C4 and C2 resulting in the formation of C3/C5 convertases (C4b,C2a) (17)(18)(19).…”

Characterization of the Interaction Between L-Ficolin/P35 and Mannan-Binding Lectin-Associated Serine Proteases-1 and -2

“…MBL circulates in association with four structurally related proteins, the MBL-associated serine proteases (MASP)-1, -2, and -3 (10 -12) and a truncated form of MASP-2 called MAp19 (19-kDa MBL-associated protein) or small MBL-associated protein (13,14). Although the precise roles of the different complexes have not yet been elucidated, it is clear that the MBL-MASP-2 complex is sufficient to trigger complement activation through cleavage of C4 and C2 (15)(16)(17).…”