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PubMed1982

Complement biosynthesis by the human hepatoma-derived cell line HepG2.

The human hepatoma-derived cell line, HepG2, synthesized and secreted functional complement proteins C1r, C1s, C2, C3, C4, C5, factor B, C1 inhibitor, C3b inactivator, a small amount of C6, and trace amounts of C8; but failed to produce detectable C1q, C7, or C9. Immunochemically, C2, C3, C4, C5, and B were isolated from culture medium as proteins with molecular sizes and subunit structures identical to the corresponding components isolated from serum. C2 and factor B from cellular lysates had slightly lower molecular weights than the corresponding proteins in culture medium. C3, C4, and C5 were detected as single chain precursor molecules in cellular lysates. These results demonstrate that human C5, like C3 and C4, is synthesized as a single chain precursor that is converted by limited proteolysis to the native two-chain molecule. It also establishes the precursor-product relationship for human pro-C4 and native C4, pro-C5, and native C5.

Carcinoma, Hepatocellular↗
PubMed1998

A monomeric human C4b-binding protein (C4bp) more efficiently inactivates C3b than natural C4bp: participation of C-terminal domains in factor I-cofactor activity.

We designed a cDNA construct encoding an artificial membrane molecule consisting of all 8 short consensus repeats (SCRs) of human monomeric C4b-binding protein (C4bp) followed by DAF's GPI anchor, named mC4bp, and expressed the protein on swine endothelial cells (SEC). At the same level of expression, mC4bp protected host cells as effectively as DAF, the most potent complement (C) regulator on the membrane. This result was unexpected from the reported functional properties of natural multimeric C4bp. Here, we investigated the mechanism whereby mC4bp has potent cell-protective activity. Our results were as follows: (1) mC4bp serves more efficiently as a methylamine-treated C3 (C3ma)-inactivating factor I-cofactor than natural C4bp and as efficiently as MCP as a methylamine-treated (C4ma)-inactivating cofactor by fluid-phase cofactor assay: (2) the potency of C3ma inactivation by mC4bp and factor I is quite high compared to those of other cofactors: (3)blocking studies using mAbs against C4bp suggested that both the 48 kDa N-terminal fragment and the C-terminal domain near the portion responsible for bundle formation participate in the high C3ma-inactivating capacity of mC4bp. Thus, acquiring high C3ma-inactivating capacity secondary to monomeric alteration leads to high C regulatory activity of mC4bp. These results infer that mC4bp differs from C4bp in its potent factor I-cofactor activity and is a good candidate as a safeguard against hyperacute rejection of xenografts.

Complement C3b Inactivator Proteins↗
PubMed2003

Effects of zinc on factor I cofactor activity of C4b-binding protein and factor H.

Complement inhibition is to a large extent achieved by proteolytic degradation of activated complement factors C3b and C4b by factor I (FI). This reaction requires a cofactor protein that binds C3b/C4b. We found that the cofactor activity of C4b-binding protein towards C4b/C3b and factor H towards C3b increase at micromolar concentrations of Zn(2+) and are abolished at 2 mM Zn(2+) and above. 65Zn(2+) bound to C3b and C4b molecules but not the cofactors or FI when they were immobilized in a native form on a nitrocellulose membrane. Zn(2+) binding constants for C3met (0.2 microM) and C4met (0.1 microM) were determined using fluorescent chelator. It appears that higher cofactor activity at low zinc concentrations is due to an increase of affinity between C4b/C3b and cofactor proteins as assessed by surface plasmon resonance. Inhibition of the reaction seen at higher concentrations is due to aggregation of C4b/C3b.

Animals↗
PubMed2005

Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases.

The complement system is pivotal in host defense but also contributes to tissue injury in several diseases. The assembly of C3 convertases (C4b2a and C3bBb) is a prerequisite for complement activation. The convertases catalyze C3b deposition on activator surfaces. Here we describe the identification of staphylococcal complement inhibitor, an excreted 9.8-kilodalton protein that blocks human complement by specific interaction with C4b2a and C3bBb. Staphylococcal complement inhibitor bound and stabilized C3 convertases, interfering with additional C3b deposition through the classical, lectin and alternative complement pathways. This led to a substantial decrease in phagocytosis and killing of Staphylococcus aureus by human neutrophils. As a highly active and small soluble protein that acts exclusively on surfaces, staphylococcal complement inhibitor may represent a promising anti-inflammatory molecule.

Bacterial Proteins↗
PubMed2004

The virulence function of Streptococcus pneumoniae surface protein A involves inhibition of complement activation and impairment of complement receptor-mediated protection.

Complement is important for elimination of invasive microbes from the host, an action achieved largely through interaction of complement-decorated pathogens with various complement receptors (CR) on phagocytes. Pneumococcal surface protein A (PspA) has been shown to interfere with complement deposition onto pneumococci, but to date the impact of PspA on CR-mediated host defense is unknown. To gauge the contribution of CRs to host defense against pneumococci and to decipher the impact of PspA on CR-dependent host defense, wild-type C57BL/6J mice and mutant mice lacking CR types 1 and 2 (CR1/2(-/-)), CR3 (CR3(-/-)), or CR4 (CR4(-/-)) were challenged with WU2, a PspA(+) capsular serotype 3 pneumococcus, and its PspA(-) mutant JY1119. Pneumococci also were used to challenge factor D-deficient (FD(-/-)), LFA-1-deficient (LFA-1(-/-)), and CD18-deficient (CD18(-/-)) mice. We found that FD(-/-), CR3(-/-), and CR4(-/-) mice had significantly decreased longevity and survival rate upon infection with WU2. In comparison, PspA(-) pneumococci were virulent only in FD(-/-) and CR1/2(-/-) mice. Normal mouse serum supported more C3 deposition on pneumococci than FD(-/-) serum, and more iC3b was deposited onto the PspA(-) than the PspA(+) strain. The combined results confirm earlier conclusions that the alternative pathway of complement activation is indispensable for innate immunity against pneumococcal infection and that PspA interferes with the protective role of the alternative pathway. Our new results suggest that complement receptors CR1/2, CR3, and CR4 all play important roles in host defense against pneumococcal infection.

Animals↗
PubMed1986

Structural analysis of human complement protein H: homology with C4b binding protein, beta 2-glycoprotein I, and the Ba fragment of B2.

We report here a partial primary structure for human complement protein H. Tryptic peptides comprising 27% of the H molecule were isolated by conventional techniques and were sequenced (333 amino acid residues). Several mixed-sequence oligonucleotide probes were constructed, based on the peptide sequence data, and were used to screen a human liver cDNA library. The largest recombinant plasmid (pH1050), which hybridized with two probes, was further characterized. The cDNA insert of this plasmid contained coding sequence (672 bp) for 224 amino acids of H. The 3' end of this clone had a polyadenylated tail preceded by a polyadenylation recognition site (ATTAAA) and a 3'-untranslated region (229 bp). Four regions of internal homology, each about 60 amino acids in length, were observed in the derived protein sequence from this cDNA clone, and a further seven from the tryptic peptide sequences. The consensus sequence for each of the repetitive units of H was four cysteines, two prolines, three glycines, one tryptophan, and two tyrosines/phenylalanines. Based on the mole percent values for each of these amino acids, it is likely that H is composed of about 20 repetitive units of this nature. Furthermore, the repetitive unit of H shows pronounced homology with the Ba fragment of B, the C4b binding protein, and beta 2-glycoprotein I. Therefore, it seems that at least portions of these proteins have evolved from a common ancestral DNA element.

Amino Acid Sequence↗
PubMed1985

Depletion of complement by light and porphyrin does not depend on sequential activation.

The mechanism of complement depletion from human serum fortified with porphyrin and irradiated with light has been reinvestigated, with the conclusion that it did not depend on the normal sequence of complement activation. Thus, disappearance of the activities of C3, C4, and C5 was not dependent on divalent cations. Purified C3-C7 were labile to porphyrin/light treatment in the absence of other components. The depletion of C4 was not prevented by potent inhibitors of C1 and, unlike the depletion of C4 seen in response to aggregated gamma globulin, was insensitive to change in temperature. Electrophoresis showed an alteration of C3 unlike that caused by cobra venom factor and that light/porphyrin treatment nonspecifically altered many serum proteins.

Complement Activation↗
PubMed1975

Interactions of C-reactive protein with the complement system. II. C-reactive protein-mediated consumption of complement by poly-L-lysine polymers and other polycations.

Cationic homopolymers of poly-L-lysine were found to activate complement (C) via C-reactive protein (CRP) and deplete C3 and C5 as well as early-acting C components. Maximum C consumption was obtained with polymers of 2,000-8,000 daltons; polymers of 1,700, 11,000, and 23,000 daltons were intermediate in reactivity, while L-lysine, lysyl-L-lysine, tetra-L-lysine, and polymers of 70,000-400,000 daltons lacked significant C-consuming activity. Naturally occurring polycations which consumed C in the presence of CRP included myelin basic proteins, cationic proteins of rabbit leukocytes, and both lysine- and arginine-rich histones; poly-L-arginine polymers of 17,000 but not 65,000 daltons also were C-consuming. Polycations without such reactivity included poly-L-orithine (5,000 and 165,000 daltons), egg white and human lysozymes, and Polybrene. The polycations which failed to induce C consumption via CRP, inhibited its consumption by both active polycations and by C-polysaccharide (CPS). The relative inhibitory capacity of phosphorylcholine and polycations in CPS- and polycations-CRP systems was consistent with the concept that phosphate esters and polycations react at the same or an overlapping combining site. The ability of certain polycations to activate C via CRP increases the potential for initiation of host reactions via C. The capacity of other polycations to inhibit C activation via CRP introduces a potential for physiologic or pharmacologic manipulation. These considerations would seem to expand the potential role of CRP in the initiation and modulation of the inflammatory response.

Animals↗
PubMed1995

Expression and distribution of cell-membrane complement regulatory glycoproteins along the human respiratory tract.

Complement in the human respiratory tract protects the host from invading microorganisms and from other inhaled insults. However, complement may also lyse the host's respiratory tract cells, leading to tissue injury. In many extrapulmonic tissues, cells express cell-membrane complement regulatory glycoproteins that protect the cells from complement-induced lysis. To determine whether these glycoproteins are expressed in human respiratory tract tissue, we studied tissue biopsies of healthy and diseased human respiratory tract from nose to alveoli for the presence of four cell-membrane complement regulatory glycoproteins (membrane cofactor protein [MCP], decay-accelerating factor [DAF], CD59, and complement receptor type 1 [CR1]) using an immunoperoxidase technique. In addition, to establish a model for in vitro studies of these glycoproteins in respiratory cells, we studied whether they are expressed in cultured nasal epithelial cells, using the same technique. Altogether, 26 tissue specimens from 22 patients were studied. We found that normal human respiratory tract from nose to alveoli express MCP, DAF, and CD59, but not CR1, and that this expression increases in inflammation and in lung cancer. In addition, expression in nasal epithelial cells is retained under cell culture conditions. These findings suggest that human respiratory tract tissue may regulate complement activation on its surface in order to avoid self-injury. We propose that imbalances in the mechanism that regulates cell-membrane complement may predispose the respiratory tract to tissue injury and disease, and that iatrogenic modulation of such imbalances may help to prevent these adverse consequences.

Adolescent↗
PubMed2000

A cluster of positively charged amino acids in the alpha-chain of C4b-binding protein (C4BP) is pivotal for the regulation of the complement system and the interaction with bacteria.

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of complement. Structural analysis of our 3D model of the C4BP alpha-chain suggested that a cluster of positively charged amino acids at the interface between complement control protein (CCP) modules 1 and 2 could be involved in ligand binding. Nine C4BP mutants, where the positively charged amino acids were changed to glutamines, were expressed. We found that all of these displayed lower apparent affinity for C4b and that the site was also a specific heparin-binding site. The mutants demonstrated decreased ability to serve as factor I cofactors in a degradation of C4b. Also, their capacity to prevent the assembly of C3-convertase and to accelerate its decay were decreased. In conclusion, we found a binding site on C4BP that is functionally important for the regulation of C3-convertase. Many strains of Streptococcus pyogenes bind C4BP, via surface M proteins, which plays an important role in pathogenesis. Using the set of C4BP mutants we identified a key recognition surface for M proteins which overlaps with the C4b-binding site. The analysis of all mutants and inhibition with monoclonal antibodies indicated that the binding sites for C4b and M proteins are only overlapping but not identical. Furthermore, we found that the molecular mechanisms involved in these two interactions differ, since the binding between M proteins and C4BP is relatively insensitive to salt in contrast to the C4BP-C4b-binding. We suggest that binding between C4b and C4BP is governed mostly by electrostatic interactions, while additional non-covalent forces cause tight binding of C4BP to streptococcal M proteins.

Amino Acids↗
PubMed1984

Characterization of the interaction of human C4b-binding protein with physiological ligands.

The binding of C4b to C4b-binding protein (C4BP) was demonstrated at physiological ionic strength by analytical ultracentrifugation. The sedimentation rate of C4BP gradually increased from 9.4 S to a maximum of 18.5 S with increasing C4b concentration. The stoichiometry of different C4BP X C4b complexes was calculated from the sedimentation-velocity data. A linear relationship was established between the number of C4b bound per C4BP and the sedimentation rate of the complex. In order to define further the C4BP-C4b interaction, sucrose density gradient ultracentrifugation was also used. Trace amounts of 125I-C4BP were centrifuged through 12 sucrose density gradients, each of which contained a different concentration of C4b throughout the gradient. The sedimentation rate of the C4BP increased with increasing C4b input to a maximum of 19.5 S. These binding data, in conjunction with the stoichiometry measurements determined in the analytical ultracentrifuge, were analyzed by the methods of Scatchard and Hill. At physiological ionic strength, C4BP exhibited four binding sites for C4b, each having an association constant of 1.2 X 10(7) M-1. A Hill coefficient of 1.1 was calculated, indicating that the four binding sites were independent. At reduced ionic strength, two additional sites were detected. The sedimentation coefficient of C4BP(C4b)6 was 24 S. The hydrodynamic data suggest that after four C4b molecules have bound to C4BP, the binding of additional C4b is sterically hindered. This interpretation implies that all six binding sites on C4BP are identical. C4BP also bound C4(H2O) (the product resulting from spontaneous hydrolysis of the thiol ester bond in native C4) and weakly bound C4c, but had no measurable affinity for native C4 or C4d at physiological ionic strength. A low-affinity interaction between C3b and C4BP was also demonstrated in the analytical ultracentrifuge. The C4BP X C3b complex was specific because C4BP mediated the cleavage of C3b by Factor I to C3bi with concomitant dissociation of the complex.

Carrier Proteins↗