Description
General Description
Native human C3 is a naturally glycosylated (~2.7%) polypeptide containing two disulfide-linked chains. C3 is central to the activation of all three pathways of complement activation (Law, S.K.A. and Reid, K.B.M. (1995)). Initiation of each pathway generates proteolytic enzyme complexes (C3 convertases) which are bound to the target surface. These enzymes cleave a peptide bond in C3 releasing the anaphylatoxin C3a and activating C3b. For a brief time (~60 µs) this nascent C3b is capable of reacting with and covalently coupling to hydroxyl groups on the target surface. Carbohydrates are the favored target, but protein hydroxyls and amino groups also react. This process of tagging the target surface with C3b is called opsonization. The reactive site in nascent C3b is a thioester (Tack B.J., et al. (1980); Pang burn M.K. and MüllerEberhard H.J. (1980)) and C3b is linked to the target through a covalent ester bond (an amide bond is formed if C3b is attached to amino groups). Most of the C3 activated during complement activation never attaches to the surface because its thioester reacts with water forming fluid phase C3b which is rapidly inactivated by factors H and I forming iC3b. Surface-bound C3b is necessary in all three pathways for efficient activation of C5 and formation of C5b-9 complexes that lyse the target cell membrane. Surface-bound C3b and its breakdown products iC3b and C3d are recognized by numerous receptors on lymphoid and phagocytic cells which use the C3b ligand to stimulate antigen presentation to cells of the adaptive immune system. The end result is an expansion of target-specific B-cell and T-cell populations. Physical Characteristics & Structure Molecular weight: 185,000 Daltons composed of two disulfide linked chains. The alpha chain is 110,000 Daltons (contains C3a and C3d domains) and the beta chain is 75,000 Daltons. Alpha and beta chains are linked through a single disulfide bond. The pI of C3 is approx. 5.9 Upon cleavage of C3 by C3 convertases, C3a (77 amino acid fragment, 9083 Da) is released from the N-terminal of the alpha chain and C3b (176,000 Da) becomes attached covalently to the surface of the activator. The crystal-derived structures of both C3 and C3b have been described (Gros, P. (2008)) and these show that large conformational changes occur in the C3b portion of C3 following cleavage of the C3aC3b peptide bond. Native C3 and C4 circulate in plasma with intramolecular thioester bonds linking a glycine and a glutamine residue in their C3d or C4d domains. These thioester bonds are susceptible to nucleophilic attack by amines such as ammonia, methylamine, hydroxylamine and hydrazine, all of which have been used to inactivate complement in serum.
CAS Number: 80295-41-6
MDL Number: MFCD00130836
Function
C3 is essential for effective complement activation and subsequent presentation of antigens to the cells of the adaptive immune system (Lambris, J.D. (1988)). Following recognition of a target complement is activated by one of the three complement pathways and enzymes (C3 convertases) are formed on the target’s surface. These enzymes (C4b,C2a or C3b,Bb) cleave C3 after Arg 77 of the alpha chain releasing the anaphylatoxin C3a and depositing C3b on the target surface. Although there is a very weak C3 bypass system that operates through the classical and lectin pathways (C4b, C2acan activate C5 without C3b at about 1/2000 the rate of C3b,C4b,C2a), C3b is necessary for effective C5 activation (Rawal N. and Pang burn M.K. (2003)). Purified C3 is extremely sensitive to freeze/thaw losing 5-10% of its activity with each freeze/thaw cycle. It is also sensitive to intermediate temperatures such as -20o C. The longer it remains at intermediate temperatures the more activity is lost. A few hours at -20o C can completely inactivate it, even though it remains completely frozen.
Assays
Complement activation requires C3. Typical assays for C3 function therefore use cell lysis endpoints in systems that lack C3 except from the source being assayed. There are three basic assays. 1) Antibody-sensitized sheep erythrocytes (EA) can be used in a CH50-type assay using C3 depleted human serum. The sensitivity of this assay is approximately 50 ng C3. 2) EA and purified components C1, C4 and C2 can be used to make EAC142 cells which utilize C3 for effective C5 activation and lysis (Dodds, A.W. and Sim, R.B. (1997)). The sensitivity of this assay is approximately 5 ng C3. 3) An alternative pathway assay may be used that employs rabbit erythrocytes and C3-depleted human serum in the presence of 5 mM MgEGTA. The sensitivity of this assay is about 200 ng C3.
In vivo
Serum concentration is 1.0 to 1.5 mg/mL with the average of 1.2 mg/mL which makes C3 the most abundant complement protein in blood. It represents approx. 2.5% of the total protein in blood and excluding albumin and immunoglobulins it is ~8% of the protein present in plasma. The primary site of synthesis is the liver, but C3 is also made in macrophages, neutrophils, astrocytes, and in endothelial and epithelial cells in many tissues of the body.
Regulation
Biosynthesis of C3 is upregulated in most cells that synthesize C3 by IL-1, IL-6, TNF-alpha, and LPS. Neutrophils are downregulated by IL-1 and IFN-gamma