Purified Proteins
| Anaphylatoxins | Depleted Sera | Antibodies | Cells & Reagents | How to Order | About Us | Contact Us | Home
Name: C9 Protein
Catalog Number: A126
Sizes Available: 250 µg/vial
Concentration: 1.0 mg/mL (see Certificate of Analysis for actual concentration)
Form: Frozen liquid
Activity: >90% versus normal human serum standard
Purity: >90% by SDS-PAGE
Buffer: 10 mM sodium phosphate, 145 mM NaCl, pH 7.3
Extinction Coeff. A280 nm = 0.988 at 1.0 mg/mL
Molecular Weight: 71,000 Da (single chain)
Preservative: None, 0.22 µm filtered
Storage: -70oC or below. Avoid freeze/thaw.
Source:
for HBsAg and for antibodies to HCV, HIV-1 and HIV-II).
Precautions: Use normal precautions for
handling human blood products.
Origin: Manufactured in the
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 C5b-8. 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 37oC 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 IFNg) 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 C5b-containing 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.
Genetics
Human chromosome location 5p 13. Accession number HSC6A. Mouse chromosome 15. Human genomic structure: the gene spans 100 kb with 11 exons.
Deficiencies
Human C9 deficiencies are quite common. A well documented study found that 1:1000 people in the Janaese population were C9 deficient although other ethnic groups have lower incidents of C9 deficiency (Horiuchi, T. et al. (1998)). Deficiencies exhibit autosomal recessive transmission. Patients generally exhibit abnormally high susceptibility to recurrent meningococcal meningitis and systemic neisserial infections. Partial deficiencies do not seem to show adverse clinical effects.
Diseases
See Deficiencies above.
Precautions/Toxicity/Hazards
This protein is purified from human plasma, 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.
Hazard
Code: B WGK
MSDS available upon request.
References
Bauer, J., Podack, E.R. and Valet, G. (1979) Determination of the number of lytic sites in biconcave and spheroid erythrocyte ghosts after complement lysis. J. Immunol. 122:2032-2036.
Dodds, A.W. and Sim, R.B. editors (1997) Complement. A Practical Approach
(ISBN 019963539)
Horiuchi, T. et al. (1998) A non-sense mutation at Arg95 is predominant in complement 9 deficiency in Japanese. J. Immunol. 160:1509-1513.
Law, S.K.A. and Reid, K.B.M. (1995) Complement 2nd
Edition (ISBN 0199633568)
Morgan, B.P. ed. (2000) Complement Methods
and Protocols. (ISBN 0-89603-654-5) Humana Press, Inc.,
Morley, B.J. and Walport, M.J.
(2000) The Complement Facts Book (ISBN 0127333606)
Academic Press,
Podack, E.R. (1984) Molecular composition of the tubular structure of the membrane attack complex of complement. J. Biol. Chem. 259: 8641-8647.
Rommel, F.A. and Mayer, M.M. (1973) Studies of guinea pig complement component C9: reaction kinetics and evidence that lysis of EAC1-8 results from a single membrane lesion caused by one molecule of C9. J. Immunol. 110:637-647.
Ross, G.D. (1986) Immunobiology of the Complement System. (ISBN 0-12-5976402) Academic Press, Orlando.
Tack, B.F., Janatova, J., Thomas,
M.L.,
FOR
RESEARCH USE ONLY.
NOT FOR HUMAN OR DRUG
USE.
Complement
Technology, Inc.
Phone: 903-581-8284
FAX: 903-581-0491
Email: contactCTI@aol.com
Web: www.ComplementTech.com