A second serine protease associated with mannan-binding lectin that activates complement

Thiel, Steffen; Vorup-Jensen, Thomas; Stover, Cordula M.; Schwaeble, Wilhelm; Laursen, Steen; Poulsen, Knud; Willis, Anthony C.; Eggleton, Paul; Hansen, Søren; Holmskov, Uffe; Reid, Kenneth B.M.; Jensenius, Jens C.

doi:10.1038/386506a0

Cited by 766 publications

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“…The concentrations of purified proteins were determined using the following absorption coefficients (A 1 cm 1% at 280 nm) and molecular weights: C1s, 14.5 and 79,800; C4, 8.3 and 205,000; C2, 8.9 and 102,000; C3, 10.0 and 185,000 (29,30,33). The absorption coefficients (A 1 cm 1% at 280 nm) used for the recombinant fragments CCP1/2-SP and CCP2-SP of MASP-2 (18.3 and 19.2, respectively) were calculated from the number of Trp, Tyr, and disulfides according to the method of Edelhoch (34), using molecular weight values of 42,710 and 35,330, calculated from the sequence (6). Because of the low amounts of material recovered, the concentrations of full-length MASP-1 and MASP-2 and of the CCP1/ 2-SP fragment of MASP-1 were estimated on the basis of Coomassie Blue staining after SDS-PAGE analysis using appropriate internal standards and respective molecular weights of 82,000, 74,200, and 45,200 (6, 9).…”

Section: Methodsmentioning

confidence: 99%

“…The plasmids containing the full-length MASP-1 and MASP-2 cDNAs were obtained as described previously (6,25). Oligonucleotides were obtained from Oligoexpress, Paris, France.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis and immunodetection of the recombinant proteins were carried out as described by Rossi et al (17), or using the ECL detection procedure of Amersham Pharmacia Biotech. The antibodies used were the mouse monoclonal anti-MASP-2 antibody 1.3B7, a rabbit polyclonal anti-MASP-2 antibody (37), and a rabbit anti-peptide antibody directed against the serine protease domain of MASP-1 (6).…”

Section: Construction Of Expression Plasmids Containing Full-length Mmentioning

confidence: 99%

“…The only available information dealing with the substrate specificity of MASP-1 and MASP-2 has been obtained on proteases isolated from human serum, and is somewhat controversial. Thus, whereas it is now widely accepted that MASP-2 cleaves both C4 and C2 (6,21,22), the observation that MASP-1 can cleave C3 and hence directly trigger complement activation (21, 23) is debated (22,24). In addition, the relative efficiency of MASP-2 with respect to C4 and C2 cleavage has not been assessed.…”

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

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

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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 ...

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Section: Methodsmentioning

confidence: 99%

“…The plasmids containing the full-length MASP-1 and MASP-2 cDNAs were obtained as described previously (6,25). Oligonucleotides were obtained from Oligoexpress, Paris, France.…”

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Expression Plasmids Containing Full-length Mmentioning

confidence: 99%

mentioning

confidence: 99%

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

Rossi¹,

Cseh²,

Bally³

et al. 2001

Journal of Biological Chemistry

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162

135

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“…The exact function of MASP-1 and MASP-3 has yet to be determined, although MASP-1 has been shown to cleave C3 weakly (12,13). MASP-2 has been shown to cleave C2 and C4 (12)(13)(14). 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.…”

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confidence: 99%

Activation of Complement Component C5

Rawal

Rajagopalan

Salvi

2008

Journal of Biological Chemistry

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Although the initiating complex of lectin pathway (called M1 in this study) generates C3/C5 convertases similar to those assembled by the initiating complex (C1) of the classical pathway, activation of complement component C5 via the lectin pathway has not been examined. In the present study kinetic analysis of lectin pathway C3/C5 convertases assembled on two surfaces (zymosan and sheep erythrocytes coated with mannan (E Man )) revealed that the convertases (ZymM1,C4b,C2a and E Man M1,C4b,C2a) exhibited a similar but weak affinity for the substrate, C5 indicated by a high K m (2.73-6.88 M). Very high affinity C5 convertases were generated when the low affinity C3/C5 convertases were allowed to deposit C3b by cleaving native C3. These C3b-containing convertases exhibited K m (0.0086 -0.0075 M) well below the normal concentration of C5 in blood (0.37 M). Although kinetic parameters, K m and k cat , of the lectin pathway C3/C5 convertases were similar to those reported for classical pathway C3/C5 convertases, studies on the ability of C4b to bind C2 indicated that every C4b deposited on zymosan or E Man was capable of forming a convertase. These findings differ from those reported for the classical pathway C3/C5 convertase, where only one of four C4b molecules deposited formed a convertase. The potential for four times more amplification via the lectin pathway than the classical pathway in the generation of C3/C5 convertases and production of pro-inflammatory products, such as C3a, C4a, and C5a, implies that activation of complement via the lectin pathway might be a more prominent contributor to the pathology of inflammatory reactions.The lectin pathway (1), a more recent discovery of complement, is activated when mannan-binding lectin (MBL), 2 a pattern-recognition molecule, binds to sugars present on the cell surface of microorganisms (2-8). In plasma, MBL circulates in complex with serine proteases called MBL-associated serine proteases (MASPs) (9 -11). We will refer to this complex as M1 in keeping with the complement nomenclature. Three types of MASPs are associated in the proenzyme form with MBL: MASP-1, MASP-2, and MASP-3. The exact function of MASP-1 and MASP-3 has yet to be determined, although MASP-1 has been shown to cleave C3 weakly (12, 13). MASP-2 has been shown to cleave C2 and C4 (12-14). 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) (17-19).C3/C5 convertases are enzymes that cleave C3 and C5. Products generated upon the cleavage of C3 and C5 (C3a, C3b, C5a, and C5b) have important biological activities (20). C3b, the larger cleavage product of C3, opsonizes bacteria, whereas C3a, the smaller product, is an anaphylatoxin with important functions such as chemotaxis of mast cells and contraction of smooth muscle cells (21-24). C5b, the larger cleavage product of C5, initiates formation ...

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