Cleavage of the fourth component of human complement (C4) by C1 esterase: Isolation and characterization of the low molecular weight product

Budzko, Delia B.; Müller‐Eberhard, Hans J.

doi:10.1016/0019-2791(70)90158-8

Cited by 59 publications

(20 citation statements)

References 21 publications

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“…The first tryptic attack is directed at the o-polypeptide chain, as is also evident from the polyacrylamide gel at a site responsible for binding of C4 onto the cell surface. Moreover, the mobility of trypsinized C4 on immunoelectrophoresis has been reported [6] to be slower than that of intact C4, an observation which is not in accordance with our result that trypsin-treated C4 had a faster P-mobility. A cathodal shift of the /3-mobility arc has been observed, however, after cleavage of C4b by C4bINA [ 181 together with the generation of a new arc of a-mobility.…”

Section: Discussioncontrasting

confidence: 99%

“…Treatment of C4 with 1% trypsin (w/w) for 1 min at 20°C totally abrogated the C4 hemolytic activity in [6]. This finding was confirmed in our study.…”

Section: Discussionsupporting

confidence: 90%

“…When activated by Cis, C4 is cleaved, releasing a small peptide, C4a, of M, -10 000 [5,6].This peptide is spasmogenic for guinea pig ileum and tachyphylactic for human C3a anaphylatoxin [7]. The rest of the molecule after Cis cleavage, i.e., C4b, can bind to the cell surface via a labile binding site.…”

Section: Methodsmentioning

confidence: 99%

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The effect of trypsin cleavage on the structure and function of human C4

et al. 1981

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Section: Discussioncontrasting

confidence: 99%

“…Treatment of C4 with 1% trypsin (w/w) for 1 min at 20°C totally abrogated the C4 hemolytic activity in [6]. This finding was confirmed in our study.…”

Section: Discussionsupporting

confidence: 90%

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The effect of trypsin cleavage on the structure and function of human C4

et al. 1981

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“…Complement proteins C2, C4, and C1 inhibitor were isolated from human plasma by means of published procedures (18 -20). For the determination of protein concentrations the following absorption coefficients (A (1%, 1 cm) at 280 nm) and molecular weights were used: C1r, 12.4 and 86,300 (21); C1s, 14.5 and 79,800 (21, 11); C1 s ␥-B, 18.3 and 47,520 (22,11); C2, 10.0 and 100,000 (18); C4, 8.2 and 205,000 (23,24); C1 inhibitor, 3.86 and 105,000 (25). The absorption coefficients (A (1%, 1 cm) at 280 nm) used for the recombinant fragments ap-SP (17.0) and CCP 2 -ap-SP (16.4) were calculated from their amino acid composition by the method of Edelhoch (26), and their molecular weights were determined by mass spectrometry analysis (see "Results").…”

Section: Methodsmentioning

confidence: 99%

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Rossi

Bally

Thielens

et al. 1998

Journal of Biological Chemistry

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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 complement. Its catalytic region (␥-B) comprises two complement control protein (CCP) modules, a short activation peptide (ap), and a serine protease domain (SP). A baculovirus-mediated expression system was used to produce recombinant truncated fragments from this region, deleted either from the first CCP module (CCP 2 -ap-SP) or from both CCP modules (ap-SP). The aglycosylated fragment CCP 2 -ap-SPag was also expressed by using tunicamycin. The fragments were produced at yields of 0.6 -3 mg/liter of culture, isolated, and characterized chemically and then tested functionally by comparison with intact C1s and its proteolytic ␥-B fragment. All recombinant fragments were expressed in a proenzyme form and cleaved by C1 r to generate active enzymes expressing esterolytic activity and reactivity toward C1 inhibitor comparable to those of intact C1 s. Likewise, the activated fragments ␥-B, CCP 2 -ap-SP, and ap-SP retained C1 s ability to cleave C2 in the fluid phase. In contrast, whereas fragment ␥-B cleaved C4 as efficiently as C1 s, the C4-cleaving activity of CCP 2 -ap-SP was greatly reduced (about 70-fold) and that of ap-SP was abolished. It is concluded that C4 cleavage involves substrate recognition sites located in both CCP modules of C1 s, whereas C2 cleavage is affected mainly by the serine protease domain. Evidence is also provided that the carbohydrate moiety linked to the second CCP module of C1 s has no significant effect on catalytic activity.The first component of complement comprises two homologous, yet distinct modular serine proteases C1r 1 and C1s assembled in the form of a Ca 2ϩ -dependent tetramer C1s-C1r-C1r-C1s. C1r is responsible for intrinsic C1 activation, a twostep process first involving autolytic C1r activation and then C1 r-mediated cleavage of proenzyme C1s. The active enzyme C1 s is a highly specific protease with trypsin-like specificity that mediates limited proteolysis of C4 and C2, the two protein substrates of C1 , thereby triggering activation of the classical pathway of complement in response to infection by various microorganisms (2-5). C4 cleavage occurs in the fluid phase and generates fragment C4b, which has the transient ability to bind covalently to the surface of the target. Cleavage of the second substrate C2, in contrast, takes place after prior formation of a C4b-C2 complex and yields C4b-C2a, the protease responsible for cleavage of complement protein C3 (5). This double proteolytic activity of C1 is controlled by C1 inhibitor, a member of the serine protease inhibitor (serpin) family, which reacts stoichiometrically with both C1 r and C1 s to form covalent protease-inhibitor complexes, resulting in disruption of the C1 s-C1 r-C1 r-C1 s tetramer (2).Human C1s is synthesized as a 673-residue single chain zymogen which, upon activation by C1 r, is cleaved between Arg 422 and Ile 423 to yield two disulfide-linked polypeptides, the N-terminal A chain ...

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“…The classical C3 convertase is a bimolecular complex of C4 and C2 (C4,2), which is formed by the proteolytic action of C1S on the precursor molecules [l]. C4 is split into two fragments, C4a and C4b, by C1 i [4,5]. The C4a fragment is released as an activation peptide from the N-terminal part of the a-chain and resembles to some extent the anaphylatoxins C3a and C5a, although it has not yet been attributed any clear biological function.…”

mentioning

confidence: 99%

Isolation of Component C4 of Human Complement and Its Polypeptide Chains

Lundwall¹,

Malmheden²,

Stålenheim³

et al. 1981

European Journal of Biochemistry

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Component C4 of human complement was purified from fresh frozen plasma with a yield of 25 x, using an initial batch separation with quaternary diethyl-(2-hydroxypropyl)aminoethyl-Sephadex followed by column chromatography on DEAE-cellulose and gel filtration in Sephadex G-200.The final product was homogenous according to polyacrylamide gel electrophoresis and immunochemical methods. Low-speed sedimentation-equilibrium analyses revealed a molecular weight of 189000, using a value of 0.736 ml/g for the partial specific volume.The polypeptide chains of reduced and alkylated C4 were separated on DEAE-Sepharose in the presence of 8 M urea. Gel filtration in Sepharose4B in 6 M guanidine hydrochloride revealed molecular weights of 88000, 72000 and 32000 for the z, / 3 and y chain respectively.The amino acid compositions of component C4 and its constitutive chains were also determined.

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Cleavage of the fourth component of human complement (C4) by C1 esterase: Isolation and characterization of the low molecular weight product

Cited by 59 publications

References 21 publications

The effect of trypsin cleavage on the structure and function of human C4

The effect of trypsin cleavage on the structure and function of human C4

Baculovirus-mediated Expression of Truncated Modular Fragments from the Catalytic Region of Human Complement Serine Protease C1s

Isolation of Component C4 of Human Complement and Its Polypeptide Chains

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