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PubMed1982

A fluorometric method for determination of C3b inactivator.

A simple method for the determination of C3b inactivator in human plasma was investigated. Fluorescent-labeled C3, which was prepared by treatment with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide, was used as the substrate. Diluted human plasma is incubated with fluorescent C3, reduced with 2-mercaptoethanol, and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The fluorescent cleavage product is then determined by scanning the gels with a fluorospectrophotometer. This method is simple and sensitive enough to determine C3b inactivator activity in 1 microliter of human plasma.

Complement C3b Inactivator Proteins↗
PubMed1990

Detection of C3bBb-stabilizing activity (C3 nephritic factor) in the serum from patients with membranoproliferative glomerulonephritis.

It is known that membranoproliferative glomerulonephritis (MPGN), hypocomplementaemia and C3 nephritic factor (C3NeF) are closely related to each other, and the presence or absence of C3NeF in the serum is important for evaluating the nature of MPGN. However, some difficulties have been encountered in detecting this factor and therefore a new assay permitting the direct detection of C3NeF without purifying IgG from the patient's serum has been devised. Using this assay method, C3bBb-stabilizing activity was observed even in sera from MPGN patients who were non-hypocomplementaemic. Furthermore, among 98 cases with hypocomplementaemia. C3NeF was found to be absent in 66 cases.

Complement C3 Convertase, Alternative Pathway↗
PubMed1987

The effect of substance(s) associated with malarial parasites on C3b inactivator.

The effects of a substance or substances associated with malarial parasites on the inhibitory role of C3b inactivator in immune adherence were investigated. The test system involved adherence of immune complexes of complement-bearing sheep erythrocytes and rabbit antibody, using human group O erythrocytes and human renal glomerular tissues as indicators. Malarial antigen and other soluble by-products of the malarial parasites, presumably present in spent culture medium, did not interfere with the inhibitory role of C3b inactivator. However, malarial pigment, both crude and purified, enhanced immune adherence in the presence of C3b inactivator. It is suggested that malarial pigment, by inhibiting the action of C3b inactivator, may play a significant role in the immunopathogenesis of renal lesions associated with malarial infections.

Animals↗
PubMed1986

Modulation of serum complement levels following exposure to polychlorinated dibenzo-p-dioxins.

Subchronic 14-day exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed serum total hemolytic complement activity (CH50) in female B6C3F1 mice at doses of 0.01, 0.05, 0.1, 0.5, 1.0, and 2.0 micrograms/kg. Serum levels of complement component C3 were also suppressed at doses of 0.5, 1.0, and 2.0 micrograms/kg. Another dioxin isomer, 1,2,3,6,7,8-hexachlorodibenzo-p-dioxin (HCDD), also produced dose-dependent suppression of complement activity at doses of 0.1, 1.0, and 10 micrograms/kg with decreased C3 levels at 10 micrograms/kg. Both TCDD and HCDD enhanced susceptibility to Streptococcus pneumoniae, a bacterial pathogen whose host defense is complement mediated. Recovery studies demonstrated that complement activity in TCDD (1 microgram/kg) and HCDD (10 micrograms/kg)-treated animals was suppressed until 50 days post-treatment, while low doses of HCDD (0.1 and 1.0 micrograms/kg) elevated CH50 levels. Acute exposure to TCDD (14 micrograms/kg) also suppressed complement CH50 and C3 levels. These studies demonstrate that the complement system and innate immunity represent potential target sites for polychlorinated dibenzo-p-dioxins.

Administration, Oral↗
PubMed1993

Associated complement C3b. Towards an understanding of its intracellular modifications.

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.

Cathepsin B↗
PubMed1986

Inhibition by lysine and arginine of the conversion of C3 and B in the serum and a purified system.

When human serum was incubated at 45 degrees C for 30 min, C3 and B were converted to C3b and Bb. Molecular weights of purified C3 and B were shown not to change after incubation at 50 degrees C. Spectropolarimetry indicated that the secondary structures of C3 and B changed after incubation at higher temperature. The titration of SH groups in the C3 molecule showed the liberation of an SH group. These results show that the alternative complement pathway is activated at raised temperatures without known activators such as zymosan or lipopolysaccharide (LPS). This may be due to the accelerated interaction of conformationally changed components of the alternative pathway such as C3 and B. Using this system, effects of various substances on the interaction of C3 and B in serum and the purified system were investigated. The addition of arginine and lysine resulted in the inhibition of the conversion of C3 and B in the serum at elevated temperature. Other amino acids such as anionic amino-acids and NaCl did not influence the conversion. In the purified system, only arginine and lysine prevented the conversion of C3 and B, when C3, B and D were incubated in the presence of Mg++ and amino-acids. Since lysine and arginine did not inhibit the enzymatic activity of D, these data suggest that arginine and lysine prevent the interaction of C3 and B in the serum at elevated temperatures.

Animals↗
PubMed1999

C1-inhibitor attenuates hyperacute rejection and inhibits complement, leukocyte and platelet activation in an ex vivo pig-to-human perfusion model.

Xenotransplantation may be a future alternative due to increased shortage of organs. Classical complement activation is central in hyperacute rejection in pig-to-human combinations. We investigated the effects of C1-inhibitor (C1-INH), a regulator of the complement and contact systems, on hyperacute rejection. Pig kidneys were perfused with fresh human blood to which either C1-INH (n = 6) or human serum albumin (n = 6) was added. The survival of the C1-INH perfused kidneys (mean 327 min) was significantly longer (p < 0.00001) than the controls (79 min). C1-INH substantially inhibited complement activation (C1rs-C1-INH complexes, C4bc, C3bc and terminal complement complex) (p < 0.001 for all) compared with the marked complement activation in the controls. No contact activation was found. Leukocytes and platelets were substantially activated (counts, myeloperoxidase, beta-thromboglobulin, thrombospondin, soluble P-selectin) in the control group, and this activation was markedly reduced by C1-INH (p < 0.02 for all). Immunohistochemistry showed less C1q, C3, TCC, IgG and fibrin deposition in the C1-INH group. C1-INH may be useful to attenuate hyperacute rejection, probably through inhibition of complement. The reduced activation of neutrophils and platelets may mainly be secondary to inhibition of complement.

Animals↗
PubMed1995

The interstitial space of the thyroid gland of marmosets (Callithrix jacchus).

The interstitial space of the thyroid gland of adult marmosets contains, like the stroma of other organs, cells and intercellular substance (matrix), blood vessels (predominantly capillaries), lymph vessels and unmyelinated nerves. It is demarcated from the follicular epithelium, the capillaries and Schwann cells by a basal lamina (BL). The perifollicular BL shows thickenings of up to 3 microns over long distances or a multilayered arrangement. These thickened segments exhibit numerous epithelial processes and ridges; in other words, the contour of the basal cell membrane is very irregular in these areas. Indentations of capillaries into the epithelium are rarely observed. The endothelium is only slightly porous. Lymph capillaries occur in large numbers. They originate freely in the interstitial space, show gaps or unspecific contacts between the thin endothelial cells; a basal lamina is missing. Bundles of 10-nm thick filaments (anchor filaments) extend to the endothelial cells of the lymph capillaries. Thin and very long (up to 8 microns) plate-like processes surround the capillaries or run parallel to the outer contour of the follicles. They originate at the poles of oval, fibroblast-like cells. Since these cells are FXIII- and C3bi-positive, they can be considered as dendritic cells. They obviously play a role in the frequently-observed autoimmune diseases of this species. In addition, monocytes and all transitional forms including macrophages, fibrocytes and lymphocytes as well as numerous mast cells occur. In the region of the BL, integrins of the beta 1-group (alpha 6) can be demonstrated immunohistologically in addition to the usual components (collagen type IV, laminin and heparan sulfate-proteoglycan). Of the fibrillar collagens type I does not occur, type III occurs only in small amounts, whereas types V and VI are observed in large amounts. The presented findings may serve as basis for more extensive experiments on these primates.

Animals↗
PubMed2004

Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase.

The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.

Animals↗
PubMed2005

Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2.

Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2).

Amino Acid Sequence↗
PubMed1999

Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.

Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.

Amino Acid Sequence↗