Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1̄s, C1̄r<sub>2</sub>–C1̄s<sub>2</sub> AND 1̄

Thielens, Nicole M.; Villiers, Marie‐Bernadette; Reboul, Anne; Villiers, Christian; Colomb, Maurice G.

doi:10.1016/0014-5793(82)80006-9

Cited by 28 publications

(15 citation statements)

References 26 publications

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“…1, lane 2) . The conversion to C2a and C2b was catalyzed by Cls at ratios as high as 5,000 (wt/wt) C2 per Cls as previously reported (20). When C2 was digested with Cls and plasmin, and analyzed by SDS-PAGE, the relative mobility of C2a was the same as that seen after digestion of Cls alone while the mobility of the C2b band was faster (Fig.…”

Section: C2 Purification and Digestion With Cls And Plasminsupporting

confidence: 82%

Angioedema induced by a peptide derived from complement component C2.

Strang

Cholin

Spragg

et al. 1988

The Journal of Experimental Medicine

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Synthetic peptides that correspond to the COOH-terminal portion of C2b enhance vascular permeability in human and guinea pig skin. In human studies, 1 nmol of the most active peptide of 25-amino acid residues produced substantial local edema. A pentapeptide and a heptapeptide corresponding to the COOH-terminal sequence of C2b each induced contraction of estrous rat uterus in the micromole range; a peptide of 25 amino acids from this region induced a like contraction of rat uterus at a concentration 20-fold lower than the smaller peptides. The vascular permeability of guinea pig skin was enhanced by doses of these synthetic peptides in a similar fashion as that observed for the concentration of rat uterus. The induction of localized edema by intradermal injection in both the guinea pig and the human proceeds in the presence of antihistaminic drugs, suggesting that there is a histamine-independent component to the observed increase in vascular permeability. Cleavage of C2 with the enzymic subcomponent of C1, C1s, yields only C2a and C2b, and no small peptides, whereas cleavage of C2 with C1s and plasmin yields a set of small peptides. These plasmin-cleaved peptides are derived from the COOH terminus of C2b, and they induce the contraction of estrous rat uterus.

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Section: C2 Purification and Digestion With Cls And Plasminsupporting

confidence: 82%

Angioedema induced by a peptide derived from complement component C2.

Strang

Cholin

Spragg

et al. 1988

The Journal of Experimental Medicine

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“…Activated C1s and complement proteins C4, C2, and C3 were purified from human plasma according to published procedures (28 -31). Partially purified C5 was prepared as described by Al Salihi et al (31), and partially purified factor B was obtained from the flowthrough fraction of the last C2 purification step on C4b-Sepharose (30). C1 inhibitor was purified from human plasma essentially as described in Ref.…”

Section: Methodsmentioning

confidence: 99%

“…Purified human ␣ 2 -macroglobulin (␣ 2 M) was kindly provided by L. Sottrup-Jensen (University of Aarhus). 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).…”

Section: Methodsmentioning

confidence: 99%

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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166

135

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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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“…C proteins C2 and C4 were isolated from human plasma by means of published procedures (24,25). The concentrations of purified proteins were determined using the following absorption coefficients (A 1%, 1 cm at 280 nm) and molecular weights: C1q, 6.8 and 459,300; C1r, 12.4 and 86,300 (8); C1r fragment CCP2-SP, 14.9 and 40,400 (23); C2, 10.0 and 100,000 (24); and C4, 8.2 and 205,000 (26,27).…”

Section: Proteinsmentioning

confidence: 99%

Functional Role of the Linker between the Complement Control Protein Modules of Complement Protease C1s

Bally¹,

Rossi²,

Thielens³

et al. 2005

The Journal of Immunology

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C1s is the modular serine protease responsible for cleavage of C4 and C2, the protein substrates of the first component of C (C1). Its catalytic domain comprises two complement control protein (CCP) modules connected by a four-residue linker Gln340-Pro-Val-Asp343 and a serine protease domain. To assess the functional role of the linker, a series of mutations were performed at positions 340–343 of human C1s, and the resulting mutants were produced using a baculovirus-mediated expression system and characterized functionally. All mutants were secreted in a proenzyme form and had a mass of 77,203–77,716 Da comparable to that of wild-type C1s, except Q340E, which had a mass of 82,008 Da, due to overglycosylation at Asn391. None of the mutations significantly altered C1s ability to assemble with C1r and C1q within C1. Whereas the other mutations had no effect on C1s activation, the Q340E mutant was totally resistant to C1r-mediated activation, both in the fluid phase and within the C1 complex. Once activated, all mutants cleaved C2 with an efficiency comparable to that of wild-type C1s. In contrast, most of the mutations resulted in a decreased C4-cleaving activity, with particularly pronounced inhibitory effects for point mutants Q340K, P341I, V342K, and D343N. Comparable effects were observed when the C4-cleaving activity of the mutants was measured inside C1. Thus, flexibility of the C1s CCP1-CCP2 linker plays no significant role in C1 assembly or C1s activation by C1r inside C1 but plays a critical role in C4 cleavage by adjusting positioning of this substrate for optimal cleavage by the C1s active site.

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Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1̄s, C1̄r₂–C1̄s₂ AND 1̄

Cited by 28 publications

References 26 publications

Angioedema induced by a peptide derived from complement component C2.

Angioedema induced by a peptide derived from complement component C2.

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

Functional Role of the Linker between the Complement Control Protein Modules of Complement Protease C1s

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Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1̄s, C1̄r2–C1̄s2 AND 1̄

Cited by 28 publications

References 26 publications

Angioedema induced by a peptide derived from complement component C2.

Angioedema induced by a peptide derived from complement component C2.

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

Functional Role of the Linker between the Complement Control Protein Modules of Complement Protease C1s

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Human complement subcomponent C2: purification and proteolytic cleavage in fluid phase by C1̄s, C1̄r₂–C1̄s₂ AND 1̄