Description
General Description
Native human C9 is a naturally glycosylated (7.8%) protein composed of a single polypeptide chain. The molecular weight is 71,000 Da. C9 binds to the C5b-8 complex
and forms the mature membrane attack complex (MAC) on cell membranes. Each pathway of complement activation generates proteolytic enzyme complexes (C3/C5
convertases) which are bound to the target surface (Ross, G.D. (1986)). 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. The C5b-8 complex is capable of
causing lysis without C9 although this is slow and requires many more complexes per cell than with C9. This property complicates C9 titrations since the precursor (C5b-8)
can also cause lysis. The primary role of C8 is to catalyze the binding of C9 and each bound C9 can bind another C9 initiating formation of a ring structure containing up to 18
molecules of C9 (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. Nevertheless, these structures are capable of causing lysis if enough are formed
in a given cell. 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 without CD59 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 ~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 C9 is 71,000 Da and it is a single polypeptide chain. The protein contains 7.8% carbohydrate attached at two N-linked glycosylation sites.
The pI of C9 is 4.7. C9 may polymerize spontaneously forming MAC rings without C5b8. The rings formed from pure C9 as well as the completed rings formed by C5b-9 with
12 to 18 C9 molecules have the unusual property of being stable in boiling SDS even though they are non-covalently bound.
CAS Number: 80295-58-5
Function
See General Description above.
Assays
Assays for C9 function are complicated by the fact that if excess C5-C8 is used cells (EA or Er) will be lysed by the C5b-8 complex. Thus it is critical to use limited C8
in these assays to keep the background lysis to a minimum. The simplest assay for C9 is to use C9-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 C9. Each unique application might require appropriate conditions to be
determined. However, a typical assay would involve mixing on wet ice ~5 µL C9-Dpl, C9-containing sample diluted with GVB++ to contain from 1 to 10 ng C9, 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 C9 or normal human serum (NHS) may be used as a
source of C9. The reaction mixture is incubated for 30 min at 37o C and 1 mL of cold GVBE added, mixed and centrifuged to spin down unlysed cells. The released
hemoglobin in the supernatant is then analyzed at 415 nm and compared to blanks without C9 (background lysis control) and cells incubated with 275 µL water instead of
GVB++ and 25 µL C9-Dpl (100% lysis control). Note as mentioned above, at inputs of serum higher than ~5 µL of C9-Dpl, EA and other target cells may also be lysed in the
absence of C9 depending on the cells’ susceptibility to C5b-9. 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 C9 is 60 µg/mL (normal range 47 to 70 µg/mL). The primary site of synthesis is the liver. C9 is also produced by monocytes,
macrophages, fibroblasts and glial cells. C9 is an acute phase protein and its synthesis is stimulated by cytokines (such as IFNγ) 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 C9 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 found bound to most soluble C5b-9
complexes. 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 proteins 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 express DAF and inactivate C3/C5 convertases and the ability express CD59 to inactivate C5b-9. This results in the spontaneous lysis by complement of the most susceptible cells
such as erythrocytes and platelets.