[3] Small-scale preparation of complement components C3 and C4

doi:10.1016/0076-6879(93)23037-N

Methods in Enzymology

Volume 223, 1993, Pages 46-61

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This chapter describes the small-scale preparation of complement components C3 and C4 proteins. The chapter discusses rapid and reproducible methods for the production of small amounts of C3 and C4. There are a number of situations when this can be useful: (1) when proteins are required only occasionally and in small amounts, for example, as hemolytic or immunochemical assay reagents, (2) when proteins are required for functional studies from small samples of plasma from individual patients or donors, (3) when proteins are required from other species, especially of small animals, where the starting volumes required for the large-scale purifications described are impractical, and (4) when recombinant proteins are recovered from tissue culture supernatants. C3 is the central and most abundant protein of the human complement system. Its intrachain thioester bond allows it to bind covalently to activating surfaces a property that it shares with C4. Two types of purification procedure are also described in the chapter: affinity methods and ion-exchange chromatography. These methods are developed, using a Pharmacia fast protein liquid chromatography (FPLC) apparatus and a Waters advanced protein purification system (APPS).

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Cited by (92)

L-ficolin-MASP arm of the complement system in schizophrenia
2023, Immunobiology
The abnormal neurodevelopment secondary to in utero adversities, such as hypoxia, malnutrition and maternal infections, underlies schizophrenia (SZ) etiology. As the genes of MBL-associated serine proteases (MASP) of the complement lectin pathway, MASP1 and MASP2, are expressed in the developing cortex and are functionally important for neuronal migration, we hypothesize that the malfunction of l-ficolin-MASP arm may also be involved in schizophrenia pathophysiology as it was shown for MBL-MASP complexes. We investigated serum l-ficolin and plasma MASP-2 levels, the activity of l-ficolin-bound MASP-2, as well as an array of the complement-related variables in chronic schizophrenic patients in the acute phase of the disease and controls without physical or mental diagnoses.
The median concentration of l-ficolin in Armenian controls was 3.66 μg/ml and similar to those reported for other Caucasian populations. SZ-cases had ∼40 % increase in serum l-ficolin (median 5.08 μg/ml; P < 0.0024). In the pooled sample, l-ficolin level was higher in males than in females (P < 0.0031), but this gender dichotomy was not affecting the variable association with schizophrenia (P < 0.016). Remarkably, MASP-2 plasma concentration showed gender-dependent significant variability in the group of patients but not in controls. When adjusted for gender and gender*diagnosis interaction, a significantly high MASP-2 level in female patients versus female controls was observed (median: 362 ng/ml versus 260 ng/ml, respectively; P < 0.0020). A significant increase in l-ficolin-bound MASP-2 activity was also observed in schizophrenia (on the median, cases vs controls: 7.60 vs 6.50 RU; P < 0.021). Correlation analyses of the levels of l-ficolin and MASP-2, l-ficolin-(MASP-2) activity and the demographic data did not show any significant association with the age of individuals, family history, age at onset and duration of the illness, and smoking. Noteworthy, the levels of l-ficolin and MASP-2 in circulation were significantly associated with the type of schizophrenia (paranoid SZ-cases had much higher l-ficolin (P < 0.0035) and lower MASP-2 levels than the other types combined (P < 0.049)). Correlations were also found between: (i) the classical pathway functional activity and l-ficolin level (rs = 0.19, P < 0.010); (ii) the alternative pathway functional activity and MASP-2 level (rs = 0.26, P < 0.00035); (iii) the activity of l-ficolin-bound MASP2 and the downstream C2 component haemolytic activity (rs = −0.19, P < 0.017); and (iv) l-ficolin and the upstream C-reactive protein (CRP) serum concentrations (r = 0.28, P < 0.018).
Overall, the results showed l-ficolin-related lectin pathway alterations in schizophrenia pathophysiology. It is likely that in addition to the MBL-MASP component over-activity reported previously, the alterations of the lectin pathway in schizophrenia also involve variations of l-ficolin-(MASP-2) on protein concentration and activity levels.
C2 by-pass: Cross-talk between the complement classical and alternative pathways
2022, Immunobiology
Citation Excerpt :
Absence of such regulatory components (C1-inhibitor, factors I and H, C4-binding protein, decay acceleration factor) is linked to uncontrolled activation of the complement cascade. Various diseases linked to the lack of appropriate regulators, or malfunction and deficiencies of soluble components of complement have been described in recent years (Lachmann, 1974; Tappeiner, 1982; Ross and Densen, 1984; Perlmutter and Colten, 1989; Atkinson, 1989; Walport and Lachmann, 1990; Densen, 1991; Whaley and Schwaeble, 1997; Frank, 2000; Botto et al., 2009; Schröder-Braunstein and Kirschfink, 2019). In general, deficiencies of the components which participate in the classical pathway of the complement system are associated with systemic lupus erythematosus (SLE) or SLE-like syndromes (Atkinson, 1989).
Several disorders associated with the total or partial absence of components of the human complement system are known. Deficiencies of classical pathway (CP) components are generally linked to systemic lupus erythematosus (SLE) or SLE-like syndromes. However, only approximately one-third of patients who lack C2 show mild symptoms of SLE. The relatively high frequency of homozygous C2 deficiency without or with minor disease manifestation suggests that there might be a compensatory mechanism which allows the activation of the CP of complement without the absolute requirement of C2. In this study we show that factor B (FB), the C2 homologue of the alternative pathway (AP) of complement, can substitute for C2. This was confirmed by using C4b as immobilised ligand and FB as analyte in Surface Plasmon Resonance (BIACORE). C2 binding to the immobilised C3b-like molecule C3(CH₃NH₂) was not seen. The estimated binding constant for C4bB complex formation was 2.00 * 10⁻⁵ [M]. We were further able to demonstrate that C4b supports the cleavage of Factor B by Factor D. Finally, cleavage of ¹²⁵I-C3 by C4bBb was evaluated and gave strong evidence that the “hybrid” convertase C4bBb can cleave and activate C3 in vitro. Cleavage activity is very low, but consistent with some of the “C2-bypass” observations of others.
Complement component C4-like protein in Atlantic cod (Gadus morhua L.) - Detection in ontogeny and identification of post-translational deimination in serum and extracellular vesicles
2019, Developmental and Comparative Immunology
The complement system is a critical part of teleost immune defences, with complement component C4 forming part of the classical and lectin pathways. Cod C4-like protein was isolated from plasma, specific antibodies generated and C4-like protein was assessed in cod sera, mucus and in extracellular vesicles (EVs) from serum and mucus. Higher levels of C4-like protein were detected in serum- than mucus-derived EVs. Post-translational deimination, caused by conversion of arginine into citrulline, can affect protein structure and function. Here we detected deiminated forms of C4-like protein in cod serum and at lower levels in mucus. C4-like protein was also found in deiminated form at low levels in EVs from both serum and mucus. C4-like protein was assessed by immunohistochemistry in cod larvae and detected in a range of organs including in liver, kidney, gut, muscle, skin and mucus, as well as in neuronal tissues of the brain, spinal cord and eye. This abundance of C4-like protein during early development may indicate roles in tissue remodelling, in addition to immune defences. The presence of deiminated C4-like protein in serum and mucosa, as well as in EVs, may suggest C4 protein moonlighting via post-translational deimination.
Expression, purification, and characterization of a human complement component C3 analog that lacks the C-terminal C345c domain
2019, Journal of Immunological Methods
Citation Excerpt :
Along these lines, the mouse monoclonal antibody WM-1 has been reported to recognize the C3c fragment of the human C3 protein (Whitehead et al., 1981). Although no high-resolution structural information is available regarding the WM-1 binding site on human C3/c, the fact that WM-1 has been used in successful affinity purification of native C3 from serum samples (Dodds, 1993) strongly suggests that the WM-1 epitope remains intact and exposed in C3/b/c. We therefore used a WM-1 derivatized SPR surface to further characterize the solution conformations of our recombinant human C3 and C3c analogs.
The complement system consists of a series of soluble and cell-surface proteins that serve numerous roles in innate immunity, development, and homeostasis. Despite its many functions, the central event in the complement system is the proteolytic activation of the 185 kDa complement component 3 (C3) into its opsonin and anaphylatoxin fragments known as C3b (175 kDa) and C3a (10 kDa), respectively. The C3 protein is comprised of thirteen separate structural domains, several of which undergo extensive structural rearrangement upon activation to C3b. In addition to this, the C-terminal C345c domain found in C3, C3b, and the terminal degradation product, C3c (135 kDa), appears to adopt multiple conformations relative to the remainder of the molecule. To facilitate various structure/function studies, we designed two C3 analogs that could be activated to a C345c-less, C3c-like state following treatment with Tobacco Etch Virus (TEV) protease. We generated stably transfected Chinese Hamster Ovary (CHO) cell lines that secrete approximately 1.5 mg of the highest-expressing C3 analog per liter of conditioned culture medium. We purified this C3 analog by sequential immobilized metal ion affinity and size exclusion chromatographies, activated the protein by digestion with TEV protease, and purified the resulting C3c analog by a final size exclusion chromatography. The conformations and activities of our C3 and C3c analogs were assessed by measuring their binding profiles to known C3/b/c ligands by surface plasmon resonance. Together, this work demonstrates the feasibility of producing a C3 analog that can be site-specifically activated by an exogenous proteolytic enzyme.
A revised mechanism for the activation of complement C3 to C3b: A molecular explanation of a disease-associated polymorphism
2015, Journal of Biological Chemistry
Citation Excerpt :
Because the C3S and C3F allotypes contain Arg102 and Gly102, respectively, we can now explain for the first time the different functionality of the C3S and C3F allotypes. C3 was purified from fresh human plasma essentially using a Q-Sepharose fast flow anion-exchange column (Amersham Biosciences) and a Mono Q 5/50 GL column (GE Healthcare) (23). The three donors were genotyped for the rs2230199 single nucleotide polymorphism (R102G) to show that all three were Arg102 (wild-type C3S allotype).
The solution structure of complement C3b is crucial for the understanding of complement activation and regulation. C3b is generated by the removal of C3a from C3. Hydrolysis of the C3 thioester produces C3u, an analog of C3b. C3b cleavage results in C3c and C3d (thioester-containing domain; TED). To resolve functional questions in relation to C3b and C3u, analytical ultracentrifugation and x-ray and neutron scattering studies were used with C3, C3b, C3u, C3c, and C3d, using the wild-type allotype with Arg¹⁰². In 50 mm NaCl buffer, atomistic scattering modeling showed that both C3b and C3u adopted a compact structure, similar to the C3b crystal structure in which its TED and macroglobulin 1 (MG1) domains were connected through the Arg¹⁰²–Glu¹⁰³² salt bridge. In physiological 137 mm NaCl, scattering modeling showed that C3b and C3u were both extended in structure, with the TED and MG1 domains now separated by up to 6 nm. The importance of the Arg¹⁰²–Glu¹⁰³² salt bridge was determined using surface plasmon resonance to monitor the binding of wild-type C3d(E1032) and mutant C3d(A1032) to immobilized C3c. The mutant did not bind, whereas the wild-type form did. The high conformational variability of TED in C3b in physiological buffer showed that C3b is more reactive than previously thought. Because the Arg¹⁰²-Glu¹⁰³² salt bridge is essential for the C3b-Factor H complex during the regulatory control of C3b, the known clinical associations of the major C3S (Arg¹⁰²) and disease-linked C3F (Gly¹⁰²) allotypes of C3b were experimentally explained for the first time.
An understanding of the solution structure of complement C3b is essential to understand its reactivity.
Ultracentrifugation and scattering revealed compact C3b structures in low salt and extended ones in physiological salt.
The two conformations reflect Arg¹⁰²–Glu¹⁰³² salt bridge formation only in low salt.
The functional differences between the major C3S (Arg¹⁰²) and C3F (Gly¹⁰²) allotypes are explained.
Zinc-induced self-association of complement C3b and factor H: Implications for inflammation and age-related macular degeneration
2013, Journal of Biological Chemistry
Citation Excerpt :
Molecular mechanisms are suggested for the initial formation of sRPEds that lead to AMD as well as an explanation for the role of zinc in reducing the occurrence of developing advanced AMD in high risk patients (38, 39). Wild-type C3 was purified from fresh human plasma by anion-exchange using a Q-Sepharose fast-flow column (Amersham Biosciences) and a Mono Q 5/50 GL column (GE Healthcare) (40). C3u was produced by incubating C3 with 200 mm hydrazine for 2 h at 37 °C in a water bath and leaving this overnight at 4 °C.
The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients.
Background: Sub-retinal pigment epithelial deposits contain complement proteins and bioavailable zinc.
Results: Ultracentrifugation and x-ray scattering show that >100 μm zinc induces oligomer formation in each of C3, C3u, and C3b, in analogy to Factor H.
Conclusion: Factor H-C3b complexes are precipitated by zinc, which inhibits complement activation.
Significance: A potential molecular mechanism for zinc-induced sub-retinal deposit formation is clarified.

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[3] Small-scale preparation of complement components C3 and C4

Publisher Summary

Mol. Immunol.

J. Immunol. Methods

Curr. Top. Microbiol. Immunol.

Curr. Top. Microbiol. Immunol.

Ann. N.Y. Acad. Sci.

Curr. Top. Microbiol. Immunol.

Annu. Rev. Biochem.

Prog. Vet. Microbiol. Immunol.

Nature (London)

Nature (London)