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PubMed1981

Deposition of C4-binding protein and beta 1H globulin in kidneys of patients with IgA nephropathy.

A study on the deposition of complement control proteins in renal tissues from patients with IgA nephropathy is described. Renal biopsy specimens were obtained from patients with IgA nephropathy and other glomerular diseases. These biopsy samples were stained with antisera to human C4-binding protein and beta 1H globulin by indirect immunofluorescent staining. It was shown that the complement system is activated in IgA nephropathy via the both alternative and classical pathways.

Carrier Proteins↗
PubMed1982

Isolation, characterization, and mechanism of action of rat beta 1H.

beta 1Hrat was purified to homogeneity from fresh rat plasma by precipitation with 28.6% ammonium sulfate followed by sequential chromatography on DEAE-Sephacel, Biorex-70, and gel filtration on Sephacryl-S300. The final material was homogeneous on SDS-PAGE analysis and had an apparent m.w. of 150,000. Reduction with dithiothreitol did not affect its m.w., suggesting that the molecule is composed of one polypeptide chain. The recovery of beta 1H was approximately 10%. A monospecific antibody against beta 1Hrat was obtained from immunized rabbits, which recognized beta 1Hrat as a protein with beta-electrophoretic mobility upon immunoelectrophoresis of fresh rat plasma. The concentration of beta 1H in plasma of normal 4-mo-old Wistar rats was 243.5 +/- 36.3 micrograms/ml (mean +/- S.D.). beta 1Hrat in this study was detected by its capacity to inhibit formation of the P-stabilized cell-bound amplification C3/C5 convertase composed of cell-bound C3bhu and Bbhu. Purified beta 1Hrat produced a dose-related, first-order loss of convertase function and release of 126I-Bbhu from the P-stabilized C3bhuBbhu convertase, indicating a mechanism of action by decay-dissociation of Bbhu from the complex C3bhuBbhuP. beta 1Hrat was at least four times less effective than beta 1Hhu in release of 125I-Bbhu from the homologous convertase composed of C3bhu and Bbhu. On the other hand beta 1H was twice as effective in releasing 125I-Bbrat from the convertase composed of C3bratBbratP when compared to beta 1Hhu. These differences are presumably dependent upon the species-specific affinity of beta 1H from humans or rats for C3bhu or C3brat, respectively.

Animals↗
PubMed1983

Surface modulation of classical pathway activation: C2 and C3 convertase formation and regulation on sheep, guinea pig, and human erythrocytes.

We examined the formation of the early classical complement (C) pathway enzymes on sheep (Es), guinea pig (Egp), and human (Eh) erythrocytes (E). Each species' E were sensitized with sufficient IgM or IgG anti-E Ab to establish equal numbers of C1-fixing sites on all E. After sensitization with 100 C1-fixing sites of Ab and excess C1, uptake of C4 was equivalent on all three cell types, judged by anti-C4 binding (for guinea pig C4) or by direct uptake of radiolabeled protein (for human C4). With equal numbers of cell-bound C1 and C4, however, there were marked differences in C2 convertase activity on Es, Egp, and Eh. Sheep EAC14 utilized C2 at least 20 times faster than Egp and Eh bearing the same number of C1 and C4 molecules. C3 cleavage was even further depressed on Egp and Eh, and was not changed by the substitution of oxyC2 for normal human C2. In whole guinea pig serum (GPS), 300 times more C1-fixing sites were required on Egp than on Es to achieve similar amounts of lysis; however, equivalent C3 uptake on Egp and Es was associated with equal extents of lysis, demonstrating that GPS lysis of these cells was regulated by early steps in classical pathway (CP) activation. Incubation of E bearing radiolabeled C4b with Factor I demonstrated that C4b on Egp was highly resistant to cleavage compared to the same protein bound to Es or Eh. Studies with partially purified C3 convertase decay-accelerating factors from Eh stroma demonstrated that these membrane proteins could not account for the observed surface regulation of CP activity because these proteins do not affect the rate of C2 cleavage by EAC14. We conclude that E surface molecules have an important role in modulation of CP activation. This surface-associated CP regulation occurs at the level of cell-bound C4b.

Animals↗
PubMed1982

The fourth component of human complement treated with amines or chaotropes or frozen-thawed (C4b-like C4): interaction with C4 binding protein and cleavage by C3b/C4b inactivator.

We have shown previously that C4 treated with amines or chaotropes, although uncleaved, exhibits properties that are similar to C4b. Studies by other groups suggest that this C4b-like form of C4 is characterized by the lack of an internal thiolester bond that is present in native C4. We report here that C4 treated with N2H4 or KSCN or frozen-thawed, unlike native C4, forms a complex with C4-binding protein (C4-bp) and is cleaved by C3b/C4b inactivator (I). Fragmentation of C4b-like C4 by I occurs without previous cleavage to C4b and requires the presence of C4-bp. Cleavage of C4b-like C4 proceeds in two steps: a small fragment (16,000 m.w.) is released first, followed by cleavage of the remaining alpha-chain fragment (83,000 m.w.) into polypeptides of 46,000 (C4d) and 32,000 m.w. All fragments, except the 46,000 m.w. fragment, are disulfide-linked to the beta- and/or gamma-chains of C4. Cleavage of C4b-like C4 probably occurs at the same points in the alp a-chain as in C4b; however, C4b-like c4 also contains C4a. Based on the m.w. determinations, the C4a portion of the alpha-chain is present in the 83,000 m.w. fragment, and after the second cleavage, in the 32,000 m.w. fragment of C4b-like C4. These findings suggest the following alignment of alpha-chain fragments: the N-terminal C4a portion is attached to a polypeptide of about 25,000 m.w. (alpha 3), which is followed in the sequence by C4d (alpha 2) (46,000 m.w.) and a small 16,000 m.w. fragment (alpha 4) that forms the C-terminus.

Amines↗
PubMed1980

Effect of histamine on monocyte complement production. II. Modulation of protein secretion, degradation and synthesis.

Using immunofluorescence and pulse-label studies with 3H-labelled amino acids, histamine was shown to inhibit the secretion of newly synthesized C2, C4, C3, factor B and beta 1H globulin by monocytes in culture. The findings suggested that protein synthesis was decreased, and that the degradation of newly synthesized intracellular protein was increased in histamine-treated monocytes. The observations that all monocytes in cultures containing histamine stained for C2, C4, and C3, factor B and beta 1H, when secretion was impaired, shows that all monocytes synthesize these proteins. These results demonstrate a negative feedback loop on C3 and C5 cleavage. The anaphylotoxins, C3a and C5a, formed as a result of C3 and C5 cleavage, release histamine from mast cells and basophils. Histamine, by inhibiting the production of C4, C2, and C3 and factor B by mononuclear phagocytes, inhibits further C3 and C5 cleavage by restricting the formation of C42, C423b and C3bBbP.

Amino Acids↗
PubMedDate not supplied

[Genetics of complement: recent aspects (author's transl)].

Genetic deficiencies of complement proteins are now more often recognized and analysed more precisely because the structure of the different complement proteins is better known. Partial defects may be detected in some components by the combined utilization of titration techniques, polymorphism studies and linkage analyses. The partial deficiency in C4 seems to be the most frequent protein deficiency in the human. The complement markers on the short arm of the sixth chromosome in man (BF, C2, C4A and C4B) are located in close proximity to the HLA-D/DR region. The combined study of the complement and HLA markers will probably allow the fine structure of the HLA region to be better defined. The association of some diseases with HLA types will probably also be better specified by the definition of associations not only with HLA-B or HLA-D types but also with the BF, C2 and C4 types.

Animals↗
PubMed1978

Capillary transport of immunoglobulins and complement proteins to the interstitial fluid and lymph.

Low concentration of immunoglobulins G, A and M and complement component proteins were found in leg lymph of normal men. The lymph/serum ratios of different proteins ranged from 0.07 to 0.24 and depended on the molecular weight of the measured protein. Major variations in concentration were found dependent on the rate of capillary filtration and lymph formation. Hemolytic activity of total complement and of the 9 components were low, with the L/S ratio from 0.06 to 0.25. The C3H50 in lymph was much lower than of the remainder of components. Remarkably low immune adherence capacity of lymph C was noted.

Capillary Permeability↗
PubMed1982

Studies on immune adherence (C3b) receptor activity of human erythrocytes: relationship between receptor activity and presence of immune complexes in serum.

Human erythrocytes (E) have surface receptors for the third component of complement (C3b-IA receptors) which mediate immune adherence haemagglutination (IAHA). We have observed that E from patients with systemic lupus erythematosus had imparied or defective C3b receptor (C3b-R) activity when circulating immune complexes (CIC) were found in serum. This phenomenon has been investigated by a newly developed method involving competitive inhibition of IAHA in patients with immune complex diseases. IAHA involving sheep E coated with antibody and complement (EAC), and indicator human E was inhibited by lysates of E with normal C3b-R activity from healthy donors and a monkey. In contrast, the lysates of E from 95% of patients bearing CIC did not inhibit IAHA, which indicated such E had defective or impaired C3b-R activity. This phenomenon was supported by control studies in which IAHA was not inhibited by lysates of E with absent, inactivated or occupied C3b-R. In those patients, in whom CIC disappeared during immunosuppressive therapy, C3b-R activity slowly returned to normal levels. Moreover, it was observed that C3b-R activity of patients' E decreased with the reappearance of CIC during exacerbations of disease. These observations suggest that CIC are carried in vivo by the C3b-R of E as well as those of the mononuclear phagocyte system, and that the E C3b-R may also contribute to the clearance of CIC.

Animals↗
PubMed1985

Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by C5b-9 does not involve increased C7 binding or cell-bound C3b.

The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.

Animals↗
PubMed2006

Structure of C3b reveals conformational changes that underlie complement activity.

Resistance to infection and clearance of cell debris in mammals depend on the activation of the complement system, which is an important component of innate and adaptive immunity. Central to the complement system is the activated form of C3, called C3b, which attaches covalently to target surfaces to amplify complement response, label cells for phagocytosis and stimulate the adaptive immune response. C3b consists of 1,560 amino-acid residues and has 12 domains. It binds various proteins and receptors to effect its functions. However, it is not known how C3 changes its conformation into C3b and thereby exposes its many binding sites. Here we present the crystal structure at 4-A resolution of the activated complement protein C3b and describe the conformational rearrangements of the 12 domains that take place upon proteolytic activation. In the activated form the thioester is fully exposed for covalent attachment to target surfaces and is more than 85 A away from the buried site in native C3 (ref. 5). Marked domain rearrangements in the alpha-chain present an altered molecular surface, exposing hidden and cryptic sites that are consistent with known putative binding sites of factor B and several complement regulators. The structural data indicate that the large conformational changes in the proteolytic activation and regulation of C3 take place mainly in the first conversion step, from C3 to C3b. These insights are important for the development of strategies to treat immune disorders that involve complement-mediated inflammation.

Binding Sites↗
PubMed1983

The role of the thioester bond in C3 and C4 in the determination of the conformational and functional states of the molecule.

The numerous ligand binding properties expressed by the activated forms of C3 (C3b) and C4 (C4b) are best explained as arising from proteolytic-cleavage-induced conformational changes. The studies described above provide direct physical evidence for such conformational transitions in both complement proteins. Significantly, however, a virtually identical conformational end state was also produced by direct nucleophilic scission of the internal thioester bond in C3 and C4 in the absence of any proteolysis. Clearly, it is the integrity of this thiolactone structure that is the determining factor in maintaining the native conformation in C3 and C4. Recent studies suggest a similar conformational role for the thioester in alpha 2-macroglobulin. Proteolytic activation in all three thioester-containing molecules renders this structure more susceptible to nucleophilic or solvolytic attack. Whether this effect is mediated by a peptide-cleavage-induced increase in the electrophilicity of the reactive carbonyl, or simply by increasing the accessibility of the solvent to this structure, is as yet unknown. In the case of C3 and C4, removal of the activation peptide also has a profound effect on the kinetics of the conformational change initiated by the loss of the thioester. This kinetic constraint has made it possible to correlate the acquisition of ligand binding properties with the spectroscopically detectable conformational changes.

Circular Dichroism↗
PubMed1987

Phagocytosis of target particles bearing C3b-IgG covalent complexes by human monocytes and polymorphonuclear leucocytes.

Immunoglobulin G (IgG) provides an efficient acceptor site for nascent C3b, and complement activation on the surface of IgG-coated bacteria has been shown to generate significant numbers of C3b-IgG complexes. We have studied the relative efficiency of IgG alone, C3b-IgG complexes, and similar densities of IgG and C3b residues deposited independently, in mediating ingestion of sheep erythrocyte (E) targets by human phagocytes. Human 125I-C3b covalently bound to rabbit anti-Forssman IgG was generated as described elsewhere (Fries et al., 1985). E,EIgMC4b, or EIgMC4b3b (prepared with IgM antibody and purified complement components) were sensitized with radiolabelled anti-Forssman IgG or C3b-IgG heterodimers to generate targets bearing IgG alone, C3b-IgG covalent complexes, or C3b and IgG in equivalent numbers but not bound to each other. Phagocytosis by monocytes and polymorphonuclear leucocytes (PMN) of targets bearing C3b-IgG was markedly enhanced relative to those bearing IgG alone, especially at levels of less than 2000 opsonin residues/target cell. Uptake of C3b-IgG-bearing targets was also significantly more resistant to competitive inhibition by ambient monomeric IgG. Phagocytosis of EIgMC4b + C3b-IgG by monocytes was superior to the uptake of either EAC4b + IgG or EAC4b3b + IgG bearing equivalent amounts of C3b and IgG not in covalent complex (P less than 0.05, n = 10). Similar results were obtained with PMN. Thus, generation of C3b-IgG complexes in vivo may not only promote complement activation and enhance C3b deposition, but also produce a compound opsonic residue which is a more potent promoter of phagocytosis than an equal number of C3b and IgG residues randomly distributed relative to each other.

Animals↗
PubMed2004

Cellular membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves C3b, an essential component of the complement system.

Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.

Blotting, Western↗
PubMed2006

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase.

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.

Animals↗