Description
General Description
Native human C7 is a naturally glycosylated protein composed of a single polypeptide chain of 92,400 Da. C7 is essential for formation of the membrane attack
complex (MAC) and is activated non-proteolytically by binding to recently-formed C5b,C6 complexes at the cell membrane. Each pathway of complement activation
generates proteolytic enzyme complexes (C3/C5 convertases) which are bound to the target surface (Ross, G.D. (1986); Law, S.K.A. and Reid, K.B.M. (1995)). These
enzymes cleave a peptide bond in the larger alpha chain of C5 releasing the anaphylatoxin C5a and activating C5b. This is the only proteolytic step in the assembly
of the C5b-9 complex. C5b is unstable, but it remains bound to the activating complex for a brief time (~2 min) during which it either binds a single C6 from the surrounding
fluid or decays and is no longer capable of forming MAC. The C5b,6 complex may also remain bound to the C3/C5 convertase where the binding of a single C7 exposes a
membrane-binding region and C5b,6,7 can partially insert into the bilipid layer of the target cell. Up to this point the complex may diffuse away from the target cell and enter
the membrane of a nearby cell. This is called bystander lysis or “reactive lysis” and can be a significant source of pathology. Each C5b-7 complex can bind one C8 protein
molecule which results in the complex inserting more firmly into the membrane. This complex binds C9 and each bound C9 can bind another C9 initiating formation of a ring
structure containing up to 18 C9 molecules (Podack, E.R. (1984)). C5b-9 complexes with one or more C9 are referred to as the Membrane Attack Complex (MAC) of
complement. Not all C5b-8 complexes have complete rings of C9 with the average being only three C9 per C5b-8 complex. Completed protein rings of C9 form the pores seen on
electron micrographs and they result in leakage of metabolites and small proteins out of the cell as well as movement of water into the cell. If sufficient numbers are inserted into
a cell membrane then water flowing into the cell, due to osmotic pressure, will rupture the cell membrane allowing the entire contents of the target cell (or a bystander cell) to be
released. Either process may result in cell death. Originally it was thought that this required only one C5b-9 complex per cell (referred to as the “one hit theory” of lysis
(Rommel F.A. and Mayer, M.M. (1973)), but this is probably not correct. For example, an erythrocyte requires ~850 C5b-9 complexes, as measured by the number of C7
molecules, for lysis to occur (Bauer, J. et al. (1979)). Host cells protected from MAC by CD59 require sufficient numbers of C5b-9 to tie up all the CD59 and then approximately
850 C5b-9 in addition. Lysis of nucleated cells requires many more C5b-9 complexes due to their size and due to the presence of multiple defense mechanisms in such cells.
Physical Characteristics & Structure
The molecular weight of C7 is 92,400 Da and it is composed of a single polypeptide chain. The pI of C7 is heterogeneous from 6.0 to 6.5.
CAS Number: 80295-57-4
Function
See General Description above.
Assays
The simplest assay for C7 is to use C7-depleted human serum and measure the lysis of EA (classical pathway) or Er (alternative pathway) as a function of the
concentration of added test sample or standard purified C7. Each unique application might require appropriate conditions to be determined. However, a typical assay would
involve mixing on wet ice 25 µL C7-Dpl, C7-containing sample diluted with GVB++ to contain from 0.5 to 5 ng C7, and sufficient GVB++ to bring the volume to 300 µL. EA
(3 X 107 cells in 200 µL) diluted in GVB++ should be added last. Purified C7 or normal human serum (NHS) may be used as a source of C7. The reaction mixture is incubated
for 30 min at 37o C and 1 mL of cold GVBE added, mixed and centrifuged to spin out unlysed cells. The released hemoglobin in the supernatant is then analyzed at 415 nm
and compared to blanks without C5 (background lysis control) and cells incubated with 275 µL water in place of GVB++ and 25 µL C7-Dpl (100% lysis control).
Many other assays have been described using EA preloaded with C1 (EAC1 cells) or preloaded with the classical pathway C5 convertase (EAC1423 cells). However, all
these assays require the use of multiple purified complement components or more difficult-to-prepare reagents (Dodds, A.W. and Sim, R.B. (1997); Morgan, B.P. (2000);
Tack, B.F., et al. (1981)).
Applications
See General Description above.
In vivo
The normal serum concentration of C7 is 56 µg/mL (normal range 49 to 70 µg/mL). The primary site of synthesis is the liver. C7 is a weak acute phase protein and
its synthesis is stimulated by the cytokines that stimulate increased biosynthesis of many other complement proteins.
Regulation
Many proteins and other components of plasma have an inhibitory effect on the lytic activity of C5b-9 complexes but there are no specific C7 inactivators. Most of the
C5b-9 inhibitors interact with the complex after the C5b-7 stage. If any of the C5bcontaining complexes fail to insert into a membrane they may self-aggregate or bind to
regulatory proteins the most prevalent of which is S Protein. S Protein (also called vitronectin) is an 80,000 Da plasma protein that binds to C5b-9 complexes that fail to
insert in the target cell membrane. This reduces damage to nearby host cells. Many other serum components inhibit or partially inhibit lysis by C5b-9 and these include SP40,40
(also known as clusterin and apolipoprotein J) and many plasma lipoprotein complexes (LDL, HDL, etc.). Host cells protect themselves from C5b-9 by a variety of mechanisms. Membrane
proteins DAF, MCP, and CR1 inhibit formation of C3/C5 convertases preventing MAC formation. CD59, also called “homologous restriction factor” and “protectin”, is a
18,000 to 20,000 Da ubiquitous component of cell membranes that is very effective at binding to and inhibiting the lytic potential of C5b-8 and C5b-9 complexes.
The species specificity of CD59 is not absolute and many mammalian CD59 do inhibit or partially inhibit MAC from other species. The specificity that is observed appears to be due to
incompatibilities between C8 of one animal and the CD59 of another. Like DAF, CD59 contains a GPI anchor (a post-translationally added lipid tail that inserts into the bilipid
layer of the cell). The disease PNH is caused by the loss of enzymes that attach the GPI tail, thus depriving cells of the ability to inactivate C3/C5 convertases and the ability to
inactivate C5b-9. This results in complement-mediated damage to and eventual lysis of long-lived blood cells such as erythrocytes and platelets.