C1r enzyme is the activated form of C1r proenzyme. C1r is a subunit of the C1 complex which is the first complement component in the classical pathway of complement. C1r proenzyme is an inactive zymogen until C1 is activated. C1r is activated when C1 binds to and is activated by antibodies bound to antigens (immune complexes) yielding C1r enzyme, the first protease that initiates the cascade. C1 is a non-covalent calcium-dependent complex of one C1q, two C1r and two C1s molecules. Each C1q binds through two or more of its six arms to the Fc domains of IgG or IgM. The binding of multiple arms to immune complexes causes the two C1r proteins in the complex (protease zymogens) to activate producing two proteases that cleave and activate the two C1s protease zymogens in the complex. The activation of C1r results from cleavage of C1r into two fragments of 57,000 and 35,000 daltons. Activation of the bound C1s molecule is the only known function of C1r enzyme. Activated C1s cleaves complement component C4 releasing C4a and initiating covalent attachment of C4b to the activating surface. Activated C1s also cleaves C2 and the larger fragment of C2 binds to the surface-attached C4b forming C4b,C2a the C3/C5 convertase of the classical pathway.
Extinction Coeff.: A280 nm = 1.15 at 1.0 mg/ml for pure C1r
Purity: >90 % by SDS PAGE (Note: C1r enzyme is 92,000 unreduced, but upon reduction runs as 57,000 and 35,000 chains on SDS PAGE)
Preservative: None, 0.22 µm filtered.
Source: Normal human serum (shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II).
Precautions: Use normal precautions for handling human blood products.
Physical Characteristics & Structure
C1r enzyme is a 92,000 dalton, two chain, trypsin-like enzyme. C1r is present in plasma at 31 μg/mL. C1r proenzyme is an unstable zymogen and it spontaneously
activates by cleaving a peptide bond in C1r producing a 57,000 dalton heavy chain and a 35,000 dalton light chain. This is the form sold as C1r enzyme. This self-activation occurs rapidly in the C1 complex upon binding to an immune complex and it occurs slowly with pure C1r. Two C1r form a C1r-C1r complex in the presence of calcium which in turn forms a stable complex with two C1s molecules in the presence of calcium.
This tetramer can exist in solution, but in the presence of C1q it binds to C1q forming the C1 complex, which is stable in the presence of calcium. C1r self-activation is controlled in part by a weak association with C1esterase inhibitor (C1-INH) when it is in the C1 complex and similar stabilization occurs with purified C1r. C1r enzyme, however, is irreversibly inactivated by binding to C1-INH.
Function
C1r enzyme can be used to activate C1s proenzyme. In the presence of calcium it is still capable of binding another C1r enzyme and two C1s molecules and this complex is capable of binding to C1q. The activated C1r enzyme will rapidly activate the two C1s proenzymes to form C1s enzymes and the resulting C1q-C1r2-C1s2 complex is a fully active C1 molecule which will activate C4 and C2 in the fluid phase or on a cell bearing antibodies, such as EA. EA are sheep erythrocytes with rabbit IgM anti-sheep erythrocytes antibodies bound to their surface.
Assays
The activity of C1r enzyme is checked by measuring its ability to bind to the protease inhibitor C1 esterase inhibitor and remain covalently bound in the presence of SDS. It may also be used to activate C1s proenzyme and to form the activated C1 complex composed of one C1q, two C1r and two C1s molecules.
Applications
See sections titled Function and Assays above.
Regulation
Activated C1r is rapidly inactivated by C1-INH . The spontaneous activation of C1r observed with pure C1 and pure C1r proenzyme is minimized by the presence of C1-INH which rapidly inactivates spontaneously activated C1r enzyme. Stabilization of the proenzyme is also due to existence of a weak complex between C1-INH and C1r proenzyme. This association apparently stabilizes C1 thus preventing spontaneous activation in serum. Separation of C1-INH from C1 during purification is one of the reasons that isolated C1 and C1r proenzyme is unstable and prone to spontaneous activation.
Genetics
The EMBL/Genbank cDNA accession number for C1r is M14058. The genes for C1r and C1s are closely linked and located on chromosome 12p13.
Deficiencies
Deficiencies of each of the three components of C1 have been found. C1r and C1s deficient patients are prone to systemic lupus erythematosus (SLE).
and recurrent pyogenic infections. They lack classical pathway function and may or may not exhibit C1r antigen in blood.
Diseases
See section titled Deficiencies above.
Precautions/Toxicity/Hazards
This protein is purified from human serum and 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.
