C3dg Quantification by PEG Precipitation and or TRIFMA.

Detection of complement activation products can be carried out in a number of ways, and different methods are used in different laboratories. No international standard for measuring complement activation in the clinical setting has been agreed upon.Here we describe a modified assay for measuring C3dg. The assay is simple, inexpensive and stable. The estimation of C3dg directly reflects complement turnover independently of activation pathway.

Optimal conditions for expressing a complement component 3b functional fragment (α2-macroglobulin receptor) gene from Epinephelus coioides in Pichia pastoris.

The α2-macroglobulin receptor (α2MR) is a major domain of complement component 3b, which may play an important role in regulating the downstream complement system in teleosts. In order to characterize the domain thoroughly larger than currently available quantities are required. Thus, in this study the Epinephelus coioides α2MR (Ec-α2MR) was expressed and secreted by the methylotrophic yeast Pichia pastoris with variations in pH and induction time to identify optimal production conditions. At pH 5.5 with 48h induction 13mg of Ec-α2MR (ca. 90% purity) was obtained from 500ml of culture. The Ec-α2MR protein product was validated by MALDI-TOF MS sequence analysis, and both Western blotting and ELISAs demonstrated that it possessed the expected activity, binding to C3b or C3b homolog antibodies, and thus can be used for future studies of the interactions and functions of complement proteins in teleosts.

Serum proteomic profiling for the early diagnosis of colorectal cancer.

No ideal serum biomarker currently exists for the early diagnosis of colorectal cancer (CRC). Magnetic bead-based fractionation coupled with MALDI-TOF MS was used to screen serum samples from CRC patients, healthy controls, and other cancer patients. A diagnostic model with five proteomic features (m/z 1778.97, 1866.16, 1934.65, 2022.46, and 4588.53) was generated using Fisher algorithm with best performance. The Fisher-based model could discriminate CRC patients from the controls with 100% (46/46) sensitivity and 100% (35/35) specificity in the training set, 95.6% (43/45) sensitivity and 83.3% (35/42) specificity in the test set. We further validated the model with 94.4% (254/269) sensitivity and 75.5% (83/110) specificity in the external independent group. In other cancers group, the Fisher-based model classified 25 of 46 samples (54.3%) as positive and the other 21 as negative. With FT-ICR-MS, the proteomic features of m/z 1778.97, 1866.16, 1934.65, and 2022.46, of which intensities decreased significantly in CRC, were identified as fragments of complement C3f. Therefore, the Fisher-based model containing five proteomic features was able to effectively differentiate CRC patients from healthy controls and other cancers with a high sensitivity and specificity, and may be CRC-specific. Serum complement C3f, which was significantly decreased in CRC group, may be relevant to the incidence of CRC.

Unique structure of iC3b resolved at a resolution of 24 Å by 3D-electron microscopy.

Activation of C3, deposition of C3b on the target surface, and subsequent amplification by formation of a C3-cleaving enzyme (C3-convertase; C3bBb) triggers the effector functions of complement that result in inflammation and cell lysis. Concurrently, surface-bound C3b is proteolyzed to iC3b by factor I and appropriate cofactors. iC3b then interacts with the complement receptors (CR) of the Ig superfamily, CR2 (CD21), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on leukocytes, down-modulating inflammation, enhancing B cell-mediated immunity, and targeting pathogens for clearance by phagocytosis. Using EM and small-angle X-ray scattering, we now present a medium-resolution structure of iC3b (24 Å). iC3b displays a unique conformation with structural features distinct from any other C3 fragment. The macroglobulin ring in iC3b is similar to that in C3b, whereas the TED (thioester-containing domain) domain and the remnants of the CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain have moved to locations more similar to where they were in native C3. A consequence of this large conformational change is the disruption of the factor B binding site, which renders iC3b unable to assemble a C3-convertase. This structural model also justifies the decreased interaction between iC3b and complement regulators and the recognition of iC3b by the CR of the Ig superfamily, CR2, CR3, and CR4. These data further illustrate the extraordinary conformational versatility of C3 to accommodate a great diversity of functional activities.

Preparation of complement fragments C3b and C3a from purified rat complement component C3 by activated cobra venom factor.

INTRODUCTION: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. METHODS: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. RESULTS: About 200 microg of CVF was purified from 100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. DISCUSSION: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact.

A simple procedure for isolation of Bufo arenarum C3 complement fraction and preparation of antiserum.

Because of the need for antibodies in our studies involving the third component of complement in Bufo arenarum, we performed a simple procedure to purify C3 from B. arenarum serum to use as antigen in the preparation of the antiserum. The strategy was based on the well-known ability of C3 to bind to zymosan (Zy), a yeast cell wall extract comprised of polysaccharides. The Zy-bound fraction showed cross reactivity with a commercial antibody to human C3 as well as a similar electrophoretic profile (SDS-PAGE) to C3 from other species. The Zy-C3 complex resulting from binding Zy to B. arenarum serum was injected into rabbits and the antiserum against this C3-like fraction was purified by protein A-Sepharose chromatography. The purified C3 antibody was found to be suitable for immunochemical studies.

Simple quantification of complement factors C3 and C3b using separation by isotachophoresis.

The separation of complement factors C3 and C3b by isotachophoresis in 1% agarose gel followed by immunoprecipitation and quantification is presented. Glycine was used as spacer in a nonequilibrium isotachophoresis (Acevedo, F., J. Chromatogr. 1991, 545, 391-396). Tricine, beta-alanine and Tris were the leading ion, terminating ion and counter ion, respectively. After electrophoresis the gel was incubated in rabbit anti-human complement factor C3c. The amounts of C3 and C3b in the sample were measured by optical densitometry of the Coomassie Brilliant blue-stained immunoprecipitates in the agarose gel. The correlation coefficient obtained for the logarithm of the integrated densitometric measurement vs. the logarithm of the amount of applied C3 was higher than 0.98 in calibration experiments. The extent of complement factor C3 activation is calculated as the ratio between the amount of C3b and the amount of C3b plus C3 and expressed as percent. The progress of complement activation from human blood plasma samples induced by Mg2+ and zymosan are presented as examples.

Identification of complement receptor type 1-related proteins on primate erythrocytes.

The purpose of this study was to characterize the structure and function of the immune adherence receptor (CR1, CD35, C3b/C4b receptor) of primates. Western blotting, immunoprecipitation, ELISA, and affinity chromatography with homologous C3b and C4b were utilized. The major cross-reactive E membrane protein of ten species of primates tested was lower in m.w. than was human CR1 and fell into two size groups of 55 to 75 and 130 to 165 kDa. There was 10- to 100-fold more CR1 per primate E than human E. Five species also expressed lesser quantities of a protein similar in m.w. (approximately 200 kDa) to human CR1. In contrast to E, the major cross-reactive protein on PBMC was similar in size to human CR1. Four species also expressed lesser amounts of a lower m.w. protein on their PBMC of the same M(r) as that found on their E. Affinity chromatography demonstrated that the approximately 200-kDa form, if present, was recovered with a similar efficiency to that of human CR1. Three patterns of binding, however, were identified among the lower m.w. proteins: 1) C3b > or = C4b; 2) C4b > C3b; and C3b only or predominantly. The fact that these E proteins cross-react with Ab to human CR1, bind homologous C3b and, in most cases, C4b, and for some species represent the only such protein expressed on their E identifies them as immune adherence receptors. The 70-kDa CR1 of the chimpanzee E seems to arise by alternative splicing of the mRNA encoding the 200-kDa protein. These data raise interesting questions relative to the evolution of CR1 in primates and provide a basis for analysis of structure-function relationships among these size forms of CR1.

Reconstitution of C5 convertase of the alternative complement pathway with isolated C3b dimer and factors B and D.

C5 convertase of the alternative complement pathway is a trimolecular complex consisting of two molecules of C3b and one molecule of Bb. We previously proposed a model of the alternative pathway C5 convertase in which the second C3b molecule binds covalently to the first C3b molecule bearing Bb, and the C5 molecule binds to each C3b molecule of the covalently linked C3b dimer, resulting in its appropriate presentation to the catalytic site on Bb. In the present study, we purified the covalently linked C3b dimer and reconstituted the C5 convertase with the C3b dimer and factors B and D to obtain evidence in support of this model. An insoluble glucan, OMZ-176, was incubated with human serum to activate the alternative pathway and to allow formation of the alternative C5 convertase on the surface of the glucan, and the glucan bearing the C5 convertase was then solubilized by incubation with glucosidases. In this way, the covalently linked C3b dimer was obtained in solution without using a detergent. The C3b dimer was then separated from enzymes, C3b monomer, C3b oligomer, and other materials by chromatographies. SDS-PAGE analysis demonstrated that the purified C3b dimer had intact alpha'-chains. Alternative pathway C5 convertase was reconstituted when the isolated C3b dimer was incubated with factors B and D. The presence of P enhanced C5 convertase formation threefold. These results support the notions that the formation of the covalently linked C3b dimer is a general phenomenon associated with activation of the alternative pathway and that the C3b dimer acts as a part of the C5 convertase.

Activation of complement by Treponema denticola.

Oral spirochetes have been shown to be associated with periodontal diseases and are present in increased numbers in lesions of greater severity. In this study, the interaction of Treponema denticola with human complement, a major antibacterial defense system, was examined. For each of two strains of T. denticola, it was found that both the classical and alternative pathways of human complement were activated in human serum upon incubation at 37 degrees C. C3 fragments were deposited on the surface of this organism following complement activation; the fragments bound included both of the major C3-derived opsonic fragments C3b and iC3b. Under incubation conditions identical to those carried out for complement activation in serum, T. denticola failed to degrade purified, hemolytically-active C3, although it readily degraded inactivated C3. Thus, despite the documented proteolytic activity of this organism, complement activation and deposition of complement-derived opsonins may be important defense mechanisms in the control of infections with T. denticola.

Immobilization of C3b with retention of functional activity.

We compared eight commercially available, pre-activated affinity chromatography supports for ability to immobilize C3b that would retain functional activity. Pre-activated supports that we studied were: cyanogen bromide activated agarose, N-hydroxysuccinimide activated agarose, Reacti-Gel HW-65, Actigel A aldehyde activated agarose, thiopropyl activated agarose, 1,4-bis(2,3-epoxypropoxy) butane activated agarose, Reacti-Gel GF-2000 and tresyl activated agarose. The amount of C3b immobilized by each support varied from 81% for Actigel A aldehyde activated agarose to only 19% for Reacti-Gel GF-2000. We examined the functional capacity of the C3b immobilized on these various supports to participate in the alternative pathway. Immobilized C3b was mixed with factors D and B of the alternative pathway and examined over time for ability to consume factor B hemolytic activity. C3b immobilized on thiopropyl activated agarose consumed factor B at a rate comparable to unbound fluid phase C3b. C3b immobilized on other supports was less active in participating in factor B consumption. Thus, we have demonstrated the ability to immobilize C3b onto a solid matrix with the immobilized C3b retaining the ability to participate in the alternative pathway. This immobilized C3b can be used to fractionate substances with high C3b binding affinity.

Localization of the covalent C3b-binding site on C4b within the complement classical pathway C5 convertase, C4b2a3b.

C5 convertase of the classical complement pathway is a trimolecular protein complex consisting of C4b, C2a, and C3b. In the complex there is an ester bond between C3b and C4b. We analyzed the C5 convertase formed on erythrocytes and localized the covalent binding site of C3b to a small region on C4b. The covalently linked C4b.C3b complex was purified from a detergent extract of the erythrocytes and digested with lysyl endopeptidase. An Mr 17,000 fragment containing the ester linkage between C4b and C3b was purified and its amino-terminal sequence was examined. Two amino acids were obtained at each cycle and identified with those in the sequences of C3 and C4. The sequence derived from C3 corresponded to the thioester region. The sequence derived from C4 started at Ala-1186. Alkali treatment of the fragment yielded an Mr 7,000 peptide derived from C4, which thus appeared to span the region of C4 from Ala-1186 to Lys-1259. Therefore, the covalent C3b-binding site on C4b is located within a 74-residue region of the primary structure. This finding supports the notion that after cleavage of C3 by the C4b2a complex, the covalent binding of metastable C3b to C4b is a specific reaction to form a trimolecular complex with a defined quaternary structure.

Discrimination between activators and nonactivators of the alternative pathway of complement: regulation via a sialic acid/polyanion binding site on factor H.

The alternative complement pathway is capable of discriminating human cells and tissues from a wide variety of potential pathogens. It has been recently demonstrated that attachment of complement component C3b to activator-derived molecules (e.g., small polysaccharides) restricts inactivation of C3b by factors H and I in a manner similar to activator surfaces. It is now shown that restriction is reversed by certain soluble polyanions (e.g., sialoglycopeptides, heparin, or dextran sulfate) that mimic the effects of sialic acid and glycosaminoglycans on human cells and tissues. Fluid-phase polyanions enhanced binding of factor H to C3b attached to activating particles, indicating that the effect resulted from increased affinity between C3b and factor H. The enhancement was specific for activator-bound C3b since no enhancement was observed on nonactivating particles. While several polyanions could cause this effect, some polyanions could not, indicating specificity. The active polyanions also inhibited lysis of cells via the alternative pathway. The binding site for sialic acid appears to reside on factor H, since factor H bound to heparin-agarose and to sialic acid-bearing fetuinagarose, whereas C3b bound to neither under the same conditions. These observations suggest that occupation of a specific site on factor H by polyanions induces an increase in the C3b-H affinity, resulting in discrimination of host cells and tissues from alternative pathway-activating foreign cells.

A new method for the estimation of C3d. Affinity clearance of C-determinant-bearing C3 molecules and fragments followed by estimation of C3d by ELISA.

A method is described to quantitate human complement fragment C3d. Test samples were treated with a predetermined excess of anti-C3c-Sepharose beads in the presence of EDTA to remove all the C-determinant-bearing C3 molecules or fragments. C3d left in the supernatant was then estimated by ELISA. Using this method, C3d could be estimated accurately in normal plasma samples. A good correlation (r = 0.93) was observed between C3d values obtained by this method and values obtained by the widely used method of Perrin and coworkers. The average C3d plasma concentration was 2.8 mg/l (SD = 0.7 mg/l, n = 21). The interassay coefficient of variation using a normal plasma pool (C3d 2.7 mg/l) was 8.3% and using normal plasma pools in which the C3d concentrations were raised to 10.3 and 17.4 mg/l by the addition of aged normal serum the levels were 8.0 and 7.5% respectively. Intra-assay coefficients of variation with these samples were 4.6, 3.0 and 2.8%, respectively. 16 patients with renal dysfunction had C3d levels in the range of 4.3-10.0 mg/l and 15 patients undergoing continued ambulant peritoneal dialysis had levels of 3.3-12.2 mg/l. The C3d content in peritoneal dialysate of patients undergoing dialysis varied from 9.3 to 383 micrograms/l.

C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.

A molecular mechanism of complement resistance of human melanoma cells.

The susceptibility of human melanoma cells to lysis by human complement after sensitization with the R24 murine IgG3 monoclonal antibody to the GD3 ganglioside antigen was investigated. It was found that the melanoma cell lines were either susceptible (greater than or equal to 70% cytotoxicity) or resistant (less than or equal to 30% cytotoxicity) to complement-mediated killing. We determined the kinetics of binding of C3 to and its subsequent fate on the melanoma cells. We found that on susceptible cell lines, maximal binding of C3 occurred within 10 min of incubation. At that time, approximately 90% of the bound C3 was in the form of C3b. During the subsequent incubation, the C3b was slowly inactivated, apparently generating the physiologic degradation products iC3b, C3dg, and C3d. However, this degradation of C3b could be inhibited without affecting the final degree of cytotoxicity, indicating that it is of no apparent consequence for the killing of susceptible melanoma cells. Very different results were obtained with resistant melanoma cells. Bound C3b was rapidly inactivated, and C3d was the predominant form of C3 on resistant cells throughout the incubation. Therefore, rapid inactivation of C3b was identified as a protective mechanism of human melanoma cells against complement attack. In addition, we found that resistance to complement is not an inherent property of the cells but depends on the antibody used for sensitization, because the resistant cell lines could be lysed after sensitization with polyclonal antiserum.

Isolation of complement-fragment-iC3b-binding proteins by affinity chromatography. The identification of p150,95 as an iC3b-binding protein.

The proteins from labelled human spleen membranes and polymorphonuclear leucocytes which bind to the iC3b fragment of complement component C3 were prepared by iC3b-Sepharose chromatography in the presence of bivalent cations. Complement receptor type 3(CR3) was eluted from iC3b-Sepharose by removal of bivalent cations. Complement receptors type 1 and 2 (present in spleen but not in polymorphonuclear leucocytes) were sequentially eluted by an NaCl gradient. An additional protein of Mr 135 000 was eluted from iC3b-Sepharose under the same conditions as those used to elute CR3. Preabsorption of the starting material on an anti-(CR3 beta-subunit) antibody column before iC3b-Sepharose chromatography removed the alpha- and beta-chains of CR3 and the 135 000-Mr protein. Preabsorption with iC3b-Sepharose before the anti-(CR3 beta-subunit) antibody column showed that iC3b binds CR3 and p150,95, the smallest member of the group of three homologous proteins that share the same beta-subunit.

Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: isolation and characterization of a biologically active fragment, C3d,g.

Limited proteolysis of C3b by C3b inactivator (factor I) consists of a two-step reaction; rapid cleavage of C3b to yield a nicked C3b derivative, iC3b, and followed by slow cleavage of iC3b to yield two antigenically distinct fragments, C3c and C3d,g. Using a fluorescence-labeled C3b as a substrate for I, we have investigated in detail the optimal conditions for the sequential cleavages of C3b by I. The pH optimum for the first cleavage was markedly affected by the ionic strength of buffers. The cleavage was maximum at pH 6.0 under physiological ionic strength but at pH 8.5 under low ionic strength (such as 1.7 mS). The second cleavage was a slow reaction and occurred only under low ionic strength and within a narrow pH range around pH 6.0. One of the products of the second cleavage, C3d,g, was isolated and shown to be a single polypeptide chain of 41,000 daltons with pI 5.0. C3d,g had leucocytosis-inducing activity, like C3d-k, which is a C3d fragment released by the action of plasma kallikrein. Trypsin digestion of C3d,g produced two fragments of 30,000 and 10,000 daltons and the 10,000-dalton fragment retained the leucocytosis inducing activity.

Classical complement pathway activation by antipneumococcal antibodies leads to covalent binding of C3b to antibody molecules.

We have examined whether or not a physical relationship exists between antipneumococcal antibodies (Ab) and C3b when Ab activate the classical complement pathway on the surface of pneumococci (Pn). After sensitization with 125I-labeled Ab, Pn were sequentially incubated with purified C1, C4, C2, and biotinylated C3. Ab molecules were then eluted from Pn, and C3b-associated molecules were purified on avidin-Sepharose. Both 125I-labeled immunoglobulin G (IgG) and [125I]IgM bound to C3b; the association was stable to incubation in 1% sodium dodecyl sulfate at 37 degrees C. The association was only partially reversed by incubation in 1 M hydroxylamine-0.5% sodium dodecyl sulfate (pH 10.5), implying that Ab and C3b were linked by amide as well as ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dithiothreitol and NH2OH eluates from the avidin-Sepharose showed that C3b bound to both heavy and light chains of the Ab. Moreover, the ability to bind to erythrocyte C3b receptors could be transferred to unsensitized Pn by eluates from Pn on which Ab had activated the classical pathway. These covalent complexes of Ab and C3b may be especially important in the opsonization of Pn by Ab and complement.