靶点/蛋白质:The gene for C4 resides on the sixth chromosome at 6p21.3. The genes for C4A and C4B are normally 10 kb apart and 21 kb long although C4B can be 21 kb or 14.6 kb long depending on the absence of a retroposon in intron 9. Both genes have 41 exons. Gene accession numbers: Human (M59815, M59816, AF019413, K02403, and U24578), Mouse (J05095 and M11729).
来源:Normal human serum (shown by certified tests to be negativefor HBsAg and for antibodies to HCV, HIV-1 and HIV-II).
存储溶液:10 mM sodium phosphate, 145 mM NaCl, pH 7.3
参考文献:References (From C3)??Dodds, A.W. and Sim, R.B. editors (1997) Complement. A Practical Approach (ISBN 019963539) Oxford University Press, Oxford. Ghannam A, et al. (2008) Human C3 deficiency associated with impairments in dendritic cell differentiation, memory B cells, and regulatory T cells. J Immunol. 181:5158-5166. Gros, P., Milder, F.J., Janssen, B.J. (2008) Complement driven by conformational changes. Nat Rev Immunol. 8:48-58. Lambris, J.D. (1988) The multifunctional role of C3, the third component of complement. Immunol Today. 9:387-93. Law, S.K.A. and Reid, K.B.M. (1995) Complement 2nd Edition (ISBN 0199633568) Oxford University Press, Oxford. Morley, B.J. and Walport, M.J. (2000) The Complement Facts Book (ISBN 0127333606) Academic Press, London. Pangburn M.K. and Müller-Eberhard H.J. (1980) Relation of putative thioester bond in C3 to activation of the alternative pathway and the binding of C3b to biological targets of complement. J Exp Med. 152:1102-14. Ross, G.D. (1986) Immunobiology of the Complement System. (ISBN 0-12-5976402) Academic Press, Orlando. Singer, L, et al., (1994) Complement C3 deficiency: human, animal, and experimental models. Pathobiology 62: 14-28. Tack BF, Harrison RA, Janatova J, Thomas ML, Prahl JW. (1980) Evidence for presence of an internal thiolester bond in third component of human complement. Proc Natl Acad Sci U S A. 77:5764-8. References (from C4)??Knutzen Steuer KL, Sloan LB, Oglesby TJ, Farries TC, Nickells MW, Densen P, Harley JB, Atkinson JP. (1989) Lysis of sensitized sheep erythrocytes in human sera deficient in the second component of complement. J Immunol. 143:2256-61. Law, S.K.A. and Dodds, A.W. (1997) Protein Sci. 6:263-274.Law, S.K.A. and Reid, K.B.M. (1995) Complement 2nd Edition (ISBN 0199633568) Oxford University Press, Oxford. May, J.E. and Frank, M.M. (1973) Hemolysis of sheep erythrocytes in guinea pig serum deficient in the fourth component of complement. I. Antibody and serum requirements. J Immunol. 111:1671-7. Morley, B.J. and Walport, M.J. (2000) The Complement Facts Book (ISBN 0127333606) Academic Press, London. Rawal N. and Pangburn M.K. (2003) Formation of high affinity C5 convertase of the classical pathway of complement. J Biol Chem. 278:38476-83. Ross, G.D. (1986) Immunobiology of the Complement System. (ISBN 0-12-5976402) Academic Press, Orlando.
简单描述:C4b is derived from native C4 upon cleavage and release of C4a. It is prepared by cleavage of purified C4 with the classical pathway activated protease C1s enzyme (#A104). Native human C4b is a glycosylated (~7.3%) polypeptide containing three disulfide-linked chains. C4b is central to the function of two of the three pathways of complement (Law, S.K.A. and Reid, K.B.M. (1995)). Both the classical and lectin pathways of complement activation generate surface-bound active proteases that cleave C4 (C1s in the classical pathway and MASP1/2 in the lectin pathway). These enzymes cleave a peptide bond in C4 releasing the anaphylatoxin C4a and activating C4b. For a fraction of a second after proteolytic activation C4b can covalently attach to the surface of the target. The thioester of metastable C4b is highly reactive and is capable of reacting with and covalently coupling C4b to amino or hydroxyl groups on the target surface. There are two variants of C4 that are common in man (C4A and C4B). Animals with only one type generally have C4B. The favored sites of attachment for variants C4A and C4B differ. Metastable C4b produced from C4A attaches primarily to amino groups while C4b produced from the C4B variant binds well to hydroxyl groups as well as amino groups (Law, S.K.A. and Dodds, A.W. (1997)). Surface-bound C4b forms the basis for formation of the C3/C5 convertase enzyme complex C4b,C2a. This enzyme activates C3, deposits C3b and thus converts itself from a weak C5 convertase to a highly efficient C5 convertase with a Km for C5 3000-fold lower than that of the C4b,C2a enzyme alone (Rawal N. and Pangburn M.K. (2003)). Surface-bound C4b is a weak opsonin and is recognized by receptors (CR1) on erythrocytes, lymphoid, and phagocytic cells. All of the complement activating functions of C4b are lost upon cleavage of the alpha chain generating C4c and C4d. The protease factor I cleaves C4b only when C4b is bound with one of the factor I cofactors: C4b binding protein (C4bBP), membrane cofactor protein (MCP) or complement receptor 1 (CR1).
Concentration: 1.0 mg/mL (see Certificate of Analysis for actual concentration)
Form: Liquid
Purity: >90% by SDS-PAGE
Extinction Coeff: A280 nm = 1.03 at 1.0 mg/mL
Molecular Weight: 176,000 Da (2 chains)
Preservative: None, 0.22 µm filtered
Physical Characteristics & Structure: C4b is a 192,000 molecular weight protein composed of three disulfide-linked chains (~88,000 Da (alpha), ~75,000 Da (beta), and ~33,000 Da (gamma)). C4 is synthesized as a single chain protein, but circulates as a 3 chain molecule of ~205,000 Da in plasma. During excretion the protein is glycosylated (6.9 %), forms an intramolecular thioester in the alpha chain and undergoes limited tyrosine sulfation. During complement activation of C4, C4a (8,757 MW) is cleaved off the N-terminal of the alpha chain producing C4b (192,000 Da). C4b retains all of the carbohydrate. Cleavage of C4b by factor I yields the soluble C4c fragment (147,000 Da) and if the C4b was attached to a surface the C4d fragment (45,000 Da) remains covalently attached to that surface. If the C4b never attached to a surface then the C4d portion is also soluble.
Function: C4b alone has no enzymatic activity. It is a structural component of the classical and lectin pathway C3 convertase (C4b,C2a) as well as a structural component of the C5 convertase of these pathways (C4b,C2a,C3b). C4b is also a ligand for complement receptor CR1.The C3 convertase of both the classical and lectin pathways (C4b,C2a) can also function as a weak C5 convertase and activate C5 without C3b at ~1/2000 the rate of C4b,C2a,C3b.
Assays: Assays of function include measurement of binding to C2a and formation of the C3 convertase and cleavage by the protease factor I in the presence of a cofactor such as C4BP or CR1. The most convenient assay measures cleavability by factor I and C4BP since it only requires C4BP and factor I and SDS gels to analyze the cleavage of the alpha chain of C4b. With C4b there are two cleavages made by factor I yielding the 45,000 Da C4d and two other fragments of ~29,000 and ~14,000 Da.
In vivo: C4b arises from the proteolytic cleavage of C4. The serum concentration of C4 is approximately 400 µg/mL. The primary site of synthesis is the liver, but C4 is also made in macrophages, momcytes, mammary gland, lung, spleen, kidney, brain, and in intestinal epithelial cells.
Regulation : C4b is regulated by both fluid phase and membrane-bound inactivators. Factor I is a serine protease that can cleave C4b at two peptide sites. Cleavage causes a structural rearrangement in C4b forming iC4b (inactive C4b) and iC4b lacks most of the binding sites that C4b possessed. Cleavage and inactivation of C4b by factor I requires that a cofactor be bound to C4b. The primary fluid phase cofactor is C4BP (225 µg/mL in plasma). Some cell membranes, such as human erythrocytes, possess CR1 which can act as a cofactor for factor I. Most human cells and tissues have MCP (membrane cofactor protein, CD46) which also acts as a cofactor for factor I. CR1 only acts on C4b on cells or immune complexes other than the cell bearing the CR1 while MCP only acts on C4b attached to the cell membrane bearing the MCP. Separate from the action of factor I the interactions of C4b with C4BP and CR1 inhibit other complement functions of C4b through competition with binding of C2a and through decay acceleration of the C3 convertase C4b,C2a. DAF (decay accelerating factor) is another membrane-bound protein that is present on most human cells and it interacts with C4b. It is not a cofactor for factor I and only promotes the dissociation of C3/C5 convertases containing C4b (C4b,C2a and C4b,C2a,C3b). The interactions of CR1, MCP, and DAF with C4b do not inactive the C4b itself and it is capable of continuing all of its complement functions once dissociated from them so long as it has not been cleaved by factor I.
Deficiencies: Complete deficiencies of C4 are rare due to the presence of four genes in most individuals . Deficient individuals have been found to lack classical pathway function and to suffer from severe immune complex disease similar to individuals with complete lack of the C4A isotype, but who possess the C4B isotype (see Diseases, below).
Diseases: Deficiency of the C4A variant in humans is correlated with an increased incidence of autoimmune disorders, primarily systemic lupus erythematosus (SLE). It is thought that the increased reactivity of C4A with amino groups found on proteins is important for effective activation of complement on immune complexes. Low concentrations of C4A due to heterozygous or homozygous states (most humans carry four C4 genes) is associated with less efficient clearance of immune complexes.
Precautions/Toxicity/Hazards: The source of this protein is human serum, therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II. MSDS available upon request.