Description
General Description
C1 is the first complement component in the cascade referred to as the classical pathway of complement. C1 binds to and is activated by antibodies bound to antigens (immune
complexes) yielding a protease that initiates the cascade. C1 is actually a non-covalent complex of three different proteins (C1q, C1r and C1s) bound together in a calcium-dependent complex.
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 is thought to introduce stress which causes the two C1r
proteins in the complex (protease zymogens) to auto-activate themselves producing two active C1r serine proteases (Morikis, D. and Lambris, J.D. (2005)). These activated C1r subunits
cleave and activate the two C1s protease zymogens in the complex. 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 which is the C3/C5 convertase of the classical pathway.
Physical Characteristics & Structure
C1 is a high molecular weight complex (766,000 Daltons) composed of one C1q molecule (410,000 Daltons), two C1r molecules (92,000 Daltons) and two C1s molecules (86,000 Daltons).
The complex of C1q, C1r and C1s is stable in the presence of calcium, but easily dissociates if calcium is removed. C1q itself is composed of 18 polypeptide chains with six each of three
different types of chains. When C1 is activated the C1r and C1s subunits are each cleaved into two chain molecules due to proteolytic activation. Thus, the SDS gel pattern of C1 is very complex.
C1 is difficult to separate from other high molecular weight complexes in serum and extensive handling results in spontaneous activation (Dodds, A.W. and Sim, R.B. (1997)). Thus,
the C1 sold by CompTech was not a pure protein. However, we have recently been successful at making a pure preparation of C1 (beginning with Lot # 17).
Function
C1 can be used to prepare cells bearing active C1 on their surface by incubating C1 with EA (Dodds, A.W. and Sim, R.B. editors (1997); Morgan, B.P. ed. (2000)). EA are sheep
erythrocytes with rabbit IgM anti-sheep erythrocytes antibodies bound to their surface (CompTech #B200). These cells (EAC1 cells) can be washed with SGVB++ buffer (see Assays
below) and they maintain bound active C1 if not washed too much or for too long. EAC14 cells may be prepared from EAC1 by addition of C4 which may subsequently be used to make
EAC142 cells by addition of C2. Similarly, EAC1 cells can be used to assay C4 or C2 (Morgan, B.P. ed. (2000)).
Assays
The unit of classical pathway activity is the CH50 and the unit of alternative pathway activity is the APH50. Similarly, the unit used to quantitate the activity of C1 is the C1H50. A
C1H50 unit is the amount of functional C1 needed to lyse 50% of 3 x 107 EA (CompTech #B200)) when that amount of C1 is incubated with 1 µg C4 and 0.2 µg C2 for 12 min at 30o
C in a total volume of 500 µL SGVB++ followed by addition of 1 mL of guinea pig serum (GPS) diluted 1:50 in GVBo containing 40 mM EDTA pH 7.2, for 30 min at 37o C. SGVB++ buffer is a
low ionic strength, slightly hypertonic buffer containing 170 mM sucrose, 0.1% gelatin, 5 mM sodium barbital, 57 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2, and 0.02% sodium azide.
Applications
See section titled Function above.
Regulation
Activated C1 is rapidly inactivated by C1-INH (C1 esterase inhibitor, CompTech #A140) In serum or plasma the spontaneous activation of C1 is minimized by the presence of C1-INH
which rapidly inactivates spontaneously activated C1r and C1s. The mechanism is thought to involve the existence of a weak complex between C1-INH and native C1 in plasma. This
association apparently stabilizes C1 thus preventing spontaneous activation (Ziccardi, R.J. (1982)). Separation of C1-INH from C1 during purification is one of the reasons that isolated C1
is unstable and prone to spontaneous activation.