Blood platelets activate the classical pathway of human complement

The assembly of the classical pathway C3 convertase in the fluid phase has been studied. The enzyme is assembled from C2 and C4 on cleavage of these proteins by C1s. Once assembled, the enzyme activity decays rapidly. Kinetic evidence has been obtained that this decay is even more rapid than previously suggested (kdecay is 2.0 min-1 at 37 degrees C). As a result, optimal C3 convertase activity is only observed with high C1s levels, which result in rapid rates of cleavage of C2 and increased rates of formation of the C3 convertase. Using high concentrations of C1s at lower temperatures (22 degrees C) in the presence of excess substrate we have demonstrated kinetically that the enzyme comprises an equimolar complex of C4b and cleaved C2. We have obtained direct evidence from gel-filtration experiments for the role of C2a as the catalytic subunit of the enzyme. C2b appears to mediate the interaction between C4 (or C4b) and C2 at pH 8.5 and at low ionic strength where the interactions can easily be detected. It may therefore be important in the assembly of the enzyme, though it is not involved in the catalytic activity. The decay of the C3 convertase reflects the release of C2a from the C4b x (C2b) x C2a complex, and the stabilizing effect of iodine on the C3 convertase is therefore apparently one of stabilizing the C4b-C2z interaction, which is otherwise weak. C1s is not a part of the C3 convertase enzyme.

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Primary structure of the A chain of human complement-classical-pathway enzyme C1r. N-terminal sequences and alignment of autolytic fragments and CNBr-cleavage peptides

Biochemical Journal ◽

10.1042/bj2250135 ◽

1985 ◽

Vol 225 (1) ◽

pp. 135-142 ◽

Cited By ~ 17

Author(s):

J Gagnon ◽

G J Arlaud

Keyword(s):

Serine Proteinase ◽

Complete Sequence ◽

Classical Pathway ◽

Human Complement ◽

Chain Fragment ◽

Single Polypeptide Chain ◽

Tryptic Cleavage ◽

Cnbr Cleavage ◽

Single Polypeptide ◽

A Chain

Activated human complement-classical-pathway enzyme C1r has previously been shown to undergo autolytic cleavages occurring in the A chain [Arlaud, Villiers, Chesne & Colomb (1980) Biochim. Biophys. Acta 616, 116-129]. Chemical analysis of the autolytic products confirms that the A chain undergoes two major cleavages, generating three fragments, which have now been isolated and characterized. The N-terminal alpha fragment (approx. 210 residues long) has a blocked N-terminus, as does the whole A chain, whereas N-terminal sequences of fragments beta and gamma (approx. 66 and 176 residues long respectively) do not, and their N-terminal sequences were determined. Fragments alpha, beta and gamma, which are not interconnected by disulphide bridges, are located in this order within C1r A chain. Fragment gamma is disulphide-linked to the B chain of C1r, which is C-terminal in the single polypeptide chain of precursor C1r. CNBr cleavage of C1r A chain yields seven major peptides, CN1b, CN4a, CN2a, CN1a, CN3, CN4b and CN2b, which were positioned in that order, on the basis of N-terminal sequences of the methionine-containing peptides generated from tryptic cleavage of the succinylated (3-carboxypropionylated) C1r A chain. About 60% of the sequence of C1r A chain (440-460 residues long) was determined, including the complete sequence of the C-terminal 95 residues. This region shows homology with the corresponding parts of plasminogen and chymotrypsinogen and, more surprisingly, with the alpha 1 chain of human haptoglobin 1-1, a serine proteinase homologue.

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In vitro inhibition of the classical pathway of human complement by a natural microbial product, colistin sulphate

Biochemical Pharmacology ◽

10.1016/0006-2952(86)90486-7 ◽

1986 ◽

Vol 35 (17) ◽

pp. 2917-2921 ◽

Cited By ~ 2

Author(s):

Syed Shafi Asghar ◽

Annemiek de Koster ◽

Hayo J. van der Helm

Keyword(s):

Classical Pathway ◽

Human Complement ◽

Microbial Product ◽

In Vitro Inhibition

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Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation

Journal of Virology ◽

10.1128/jvi.01935-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3283-3294 ◽

Cited By ~ 30

Author(s):

Viveka Nand Yadav ◽

Kalyani Pyaram ◽

Jayati Mullick ◽

Arvind Sahu

Keyword(s):

Hot Spots ◽

Classical Pathway ◽

Variola Virus ◽

Amino Acid Residues ◽

Human Complement ◽

Binding Ability ◽

Complement Control Protein ◽

Functional Advantage ◽

Complement Regulator ◽

Control Protein

ABSTRACT Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

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Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP

Infection and Immunity ◽

10.1128/iai.00279-10 ◽

2010 ◽

Vol 78 (7) ◽

pp. 3207-3216 ◽

Cited By ~ 53

Author(s):

Angela S. Barbosa ◽

Denize Monaris ◽

Ludmila B. Silva ◽

Zenaide M. Morais ◽

Sílvio A. Vasconcellos ◽

...

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Functional Characterization ◽

Lectin Pathway ◽

Classical Pathway ◽

Dependent Manner ◽

Human Complement ◽

Cofactor Activity ◽

Complement Regulator

ABSTRACT We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis.

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A novel human complement-related protein, C1r-like protease (C1r-LP), specifically cleaves pro-C1s

Biochemical Journal ◽

10.1042/bj20041196 ◽

2005 ◽

Vol 387 (1) ◽

pp. 165-173 ◽

Cited By ~ 9

Author(s):

Christina LIGOUDISTIANOU ◽

Yuanyuan XU ◽

Gerard GARNIER ◽

Antonella CIRCOLO ◽

John E. VOLANAKIS

Keyword(s):

Serine Proteases ◽

Catalytic Efficiency ◽

Structural Features ◽

Large Deletion ◽

Northern Blotting ◽

Enzymic Activity ◽

Classical Pathway ◽

Protease Gene ◽

Human Complement ◽

Serine Protease Domain

The availability of the human genome sequence allowed us to identify a human complement-related, C1r-like protease gene (c1r-LP) located 2 kb centromeric of the C1r gene (c1r). Compared with c1r, c1r-LP carries a large deletion corresponding to exons 4–8 of c1r. The open reading frame of the C1r-LP cDNA predicts a 50 kDa modular protein displaying 52% amino acid residue identity with the corresponding regions of C1r and 75% identity with a previously described murine C1r-LP. The serine protease domain of C1r-LP, despite an overall similarity with the AGY group of complement serine proteases, has certain structural features characteristic of C2 and factor B, thus raising interesting evolutionary questions. Northern blotting demonstrated the expression of C1r-LP mRNA mainly in the liver and ELISA demonstrated the presence of the protein in human serum at a concentration of 5.5±0.9 μg/ml. Immunoprecipitation experiments failed to demonstrate an association of C1r-LP with the C1 complex in serum. Recombinant C1r-LP exhibits esterolytic activity against peptide thioesters with arginine at the P1 position, but its catalytic efficiency (kcat/Km) is lower than that of C1r and C1s. The enzymic activity of C1r-LP is inhibited by di-isopropyl fluorophosphate and also by C1 inhibitor, which forms stable complexes with the protease. Most importantly, C1r-LP also expresses proteolytic activity, cleaving pro-C1s into two fragments of sizes identical with those of the two chains of active C1s. Thus C1r-LP may provide a novel means for the formation of the classical pathway C3/C5 convertase.

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