Name: Factor H
SKU: A137C
Price: 120.00 USD
Availability: InStock

Description

General Description
Complement factor H (fH) is purified from normal human serum. It is an essential regulatory component of the alternative pathway of complement. It is critical
for prevention of complement activation on host cells and tissues, especially the kidney. It has two functional activities: 1) it controls the formation and decay of the alternative
pathway C3/C5 convertase (decay accelerating activity) and 2) it acts as a cofactor for factor I which proteolytically inactivates C3b when C3b is bound to factor H (cofactor activity).
Factor H is a 155,000 Da protein composed of 20 homologous domains arranged like beads on a semi-flexible string. The N-terminal 5 domains bind to C3b and inhibit
binding of factor B thus reducing the formation of C3/C5 convertase. Factor H also binds to preformed C3/C5 convertases (C3b,Bb and C3b,Bb,C3b) and causes rapid release of
the catalytic subunit Bb (decay acceleration). These activities are essential for controlling the spontaneous activation of the alternative pathway amplification process in
plasma. In addition, factor H controls the formation and decay of these enzymes when C3b is attached to the surface of particles. It is most effective on host cells and less
effective on foreign particles for reasons described below. The alternative pathway of complement is constantly activating by “kickover” producing fluid phase C3b-like C3(H2O) and C3b.
Factor H can bind to these proteins and act as a cofactor so that factor I (a serine protease that circulates in active form) can cleave their alpha chains producing inactive
proteins (iC3b or iC3(H2O)). If C3b is not inactivated in this way it continues to form C3 convertases and consumes factor B and C3. If C3b is attached to surfaces
it is converted to iC3b by factors H and I in a similar manner. Factor H is more effective when C3b resides on a host cell due to the presence of host markers recognized by factor H.
Complement-mediated damage to the host is minimized due to host specific recognition by factor H. Factor H appears to regulate discrimination between potential pathogens and host
cells and tissues by recognizing host markers. C3b attached to a surface can initiate the amplification cascade of the alternative pathway. Factor H prevents this on host cells and
allows it to occur on surfaces that do not bear host-like markers. These host-specific structures are thought to be polyanionic clusters such as sialic acids and sulfated
glycosaminoglycans. Recognition of host markers occurs through multiple polyanion binding sites located in domains 6-20 of factor H. One site is located in domain 7 and a
mutation in this domain (Y402H) is strongly associated with complement activation and tissue destruction in age-related macular degeneration (Zipfel, P.F. et al. (2006)). A
tentative site is located in the domain 12-14 region and a very important site is located at the C-terminal in domains 19-20. This C-terminal site appears to be the main site that
aids binding to host surfaces. Mutations affecting or located in these domains lead to activation of the alternative pathway of complement in inherited hemolytic uremic
syndrome (Zipfel, P.F. et al. (2006)). This site appears to be the site involved in polyanion-dependent dimer and tetramer formation of factor H (Pang burn, M.K. et al. (2009)).

Physical Characteristics & Structure
Molecular weight: 155,000 Da. Factor H is a heavily glycosylated (16%) protein composed of a single polypeptide chain. The pI = 5.4 – 6.0. The structure consists of 20
homologous domains of approximately 60 amino acids each connected by short spacers of 3 to 8 amino acids. Some interdomain linkages appear to be highly flexible and some
appear to be rigid. Each domain has two disulfides and numerous invariant amino acids. These domains are referred to as CCP (Complement Control Protein domains) or SCR
(Short Consensus Repeat domains) although CCP has recently gained wider acceptance. Both NMR- and crystal-derived structures have been published for various domains. The
domains are ellipsoid and the overall length of factor H has been estimated to be between 40 and 80 nm.

Function
See General Description above.

Assays
Functional assays of factor H measure either its decay accelerating activity or its factor I cofactor activity. The most convenient cofactor assay measures the cleavage of
purified C3b by SDS gels. This must be done in the presence of factor I (Morgan, B.P. (2000)). A typical C3b cleavage assay should contain approximately 4 µg C3b, various
amounts of factor H from 0.1 to 1 µg and 1 µg factor I in a total volume of 15 µL. The assays should be set up on wet ice, then incubated for 10 min at 37o C at which time SDS
sample buffer containing reducing agent should be added to the tubes and the samples heated for 5 min. SDS PAGE gels run under reducing conditions should reveal cleavage
of the alpha chain of C3b. A continuously monitored fluorescent assay has been reported (Pang burn, M.K. et al. (1983)) which takes advantage of the approximately 8-fold drop in
fluorescence of ANS (8-anilino-1-naphthalenesulfonic acid) in the presence of C3b when that C3b is converted to iC3b. Decay acceleration is more difficult to assay because it requires
the measurement of the increased rate of decay of the C3 convertase C3b,Bb in the presence of factor H. C3b,Bb has a natural half life at 37o C of 90 seconds, but by substituting Ni++ for Mg++
ions and working at room temperature, the half life can be increased to about 20 min. Two assays may be used: 1) lysis of sheep erythrocytes coated with C3b,Bb (Dodds,
A.W. and Sim, R.B. (1997)) or 2) zymosan or erythrocytes coated with C3b,Bb made with 125I-B (Pang burn, M.K., et al. (2000)). In the EsC3b,Bb lysis assay, the higher the
factor H concentration during the preincubation period the less lysis is seen in the second step where lysis occurs in the presence of EDTA and the terminal pathway proteins
(Dodds, A.W. and Sim, R.B. (1997)). In the assays using ZymC3b,Bb with radiolabeled factor B, a ten minute incubation followed by a rapid wash of the cells results in less
radioactivity on the cells incubated in the presence of factor H than on the cells in the absence of factor H.

Applications
Purified factor H as well as whole plasma from normal donors has been used as replacement therapy in inherited HUS. This treatment is effective but temporary.

In vivo
The serum concentration is generally accepted as 500 µg /mL, however, literature values for the extinction coefficient vary from 0.9 to 1.95 A280 nm for a 1 mg/mL solution
(Pang burn, M.K. et al. (2009)) and estimates of plasma concentrations range from 250 to 564 µg/ml. The primary site of synthesis is the liver. In addition, synthesis has been
demonstrated in monocytes, endothelial cells, fibroblasts, platelets, glial cells and myoblasts. In these cells and in liver synthesis is increased by IFN gamma.

Regulation
IFN gamma has been shown to increase liver synthesis of factor H and a similar effect is seen on synthesis by monocytes, endothelial cells, fibroblasts and myoblasts.

Specifications

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: >95% by SDS-PAGE
Buffer: 10 mM sodium phosphate, 145 mM NaCl, pH 7.3
Extinction Coeff.: A280 nm = 1.24 at 1.0 mg/mL
Molecular weight: 155,000 Da (single chain)
Preservative: None, 0.22 µm filtered
Storage: -70o C or below. Avoid freeze/thaw.
Source: Normal human serum (shown by certified tests to be negative 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 USA.

Safety and Handling

Precautions/Toxicity/Hazards
This protein is purified from pooled human serum, 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 and for antibodies to HCV, HIV-1 and HIV-II.
Hazard Code: B WGK Germany 3
MSDS available upon request.
CAS Number: 80295-65-4

Additional Information

Genetics
The human chromosome location is 1q32 while the mouse is 1,74.1 cM. It spans 22 exons. Accession numbers: human Y00716 and mouse M12660

Deficiencies
Human factor H deficiencies are rare and usually result in pathology. In the absence of factor H there is uncontrolled activation of the alternative pathway of
complement. This results in low C3 and factor B levels. Patients suffer from renal disease, infections especially meningococcal infections, glomerulonephritis, systemic
lupus erythematosus and are chronically hypocomplementemia. In factor H knock-out mice the primary cause of death is kidney failure due to massive uncontrolled
complement activation.

Diseases
Surprisingly, the complement field was known for years as an immune system without a disease. By this it was meant that although complement seemed to be involved
in most diseases where the immune system was activated, no diseases had been shown to be due to complement itself and there were questions about its importance to disease
processes where it was involved, such a SLE. Then multiple examples of the failure of natural complement control systems began to reveal the destructive role complement
played in many diseases. The first example was PNH (paroxysmal nocturnal hemoglobinuria) where an acquired inability to attach lipid anchors to two membrane bound
regulatory proteins (DAF and CD59) resulted in human erythrocytes which were attacked by complement and lysed in 3 to 6 days instead of circulating for 60 to 90 days
(Nicholson-Weller , A., et al. (1983); Pang burn, M.K., et al. (1983); Brodsky, R.A. (2008)). Even though factor H protects these cells allowing extended time in circulation
(Ferreira, V.P. and Pang burn, M.K. (2007)) it cannot overcome the absence of the two cell-bound regulators. Atypical hemolytic uremic syndrome (aHUS) was shown
to be associated with mutations affecting the C-terminal of factor H (Zipfel, 2006). As described above in the General Description, this region of factor H binds factor H
to host surfaces and to deposited C3b. This two-site attachment at the C-terminal (domains 19-20) anchors factor H to the surface which allows the N-terminal regulatory
domains to inhibit further activation at that site. Mutations that reduce the effectiveness of this anchor lead to reduced control of alternative pathway activation on host tissues.
Because control of spontaneous fluid phase activation by the n-terminal domains is intact serum from affected individuals contains a fully functional alternative pathway that cannot be
adequately controlled on surfaces. Thus, aHUS is characterized by spontaneous complement activation that destroys the kidney. In 2005 four research groups using the first data
from the human genome project simultaneously identified a strong link between age-related macular degeneration and a single amino acid change in domain 7 of factor H
(de Córdoba, S.R. and de Jorge, E.G. (2008)). This region is known to contain a high affinity polyanion binding site and by a mechanism that is still unclear this appears to lead
to complement activation primarily on retinal tissue late in life.