Complement System Proteins

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 Complement System Proteins Background

Components and the functions of the complement system
Complement system was first discovered as the heat-sensitive effector component of serum in the late 19th century. The complement system consists of more than 30 proteins that are either soluble or membrane-associated. Soluble complement proteins account for about 5% of the total human blood plasma proteins. Besides blood plasma, soluble complement proteins are also present in other body fluids, although at a comparably low level. Membrane associated complement proteins consist of complement receptors and complement regulatory proteins.
Complement system plays a critical role in both innate and adaptive immune responses. The functions of the complement system include: 1) lysis of pathogens and foreign cells, 2) opsonization of the pathogens leading to the phagocytosis, 3) chemotaxis of phagocytes to the site of infection by generating anaphylatoxins, 4) clearance of the harmful antigen-antibody complexes, 5) B cell activation and differentiation, 6) modulation of T cell mediated-immune response.
Complement functions to fight infection through three primary mechanisms: 1) opsonization, or coating, of pathogens so that they can be recognized by cells of the immune system, 2) production of chemoattractants and inflammatory mediators that recruit immune cells to the site of infection and modulate the immune response, 3) direct lysis of bacteria by disrupting their cell membrane. The 35 known complement proteins function as part of an extra-cellular signaling cascade, in which most of the signals are transmitted via enzymatic cleavage of the proteins. The complement system is activated by various pathogenic particles via three known pathways - the classical pathway, the alternative pathway, and the lectin pathway.

Activation pathways of complement system
Activation of the classical pathway begins with C1q, the protein that shares structural homology to SP-A and MBL. C1q is associated with two other proteins C1r and C1s that come together to form a proteolytic enzyme complex when C1q binds to antibody on the surface of a pathogen. This complex, called active C1, recruits C4, the next component of the classical pathway, to the pathogen surface. Upon binding, C4 is enzymatically cleaved to form C4b, which is covalently bound to the pathogen surface, and C4a, which is released from the pathogen. C4b is able to recruit the next complement component, C2, which is cleaved by active C1 to form C2a and C2b. C2a joins C4b to form a new enzyme complex on the pathogen surface; C2b is released. This complex, C4b2a, is known as the classical C3 convertase and is able to recruit and cleave C3, bringing C3b to the pathogen surface and releasing C3a. C3 is the most abundant and the most important complement protein. All three pathways of complement activation converge at C3.
Activation of the alternative pathway depends on spontaneous hydrolysis of an internal thioester bond of C3, resulting in the formation of C3H2O. C3H20 recruits factor B and Factor D to form C3H20Bb, an enzyme complex which is able to cleave C3. This process allows for slow, continuous cleavage of C3 in the blood to create low levels of C3b. These products are quickly degraded under normal conditions, but If C3b is formed near the surface of bacteria, it can bind covalently to the surface. In the presence of a bacterial surface, continued activation of the alternative pathway is favored. Surface bound C3b can recruit Factor B, which allows Factor D to catalyze a change in Factor B. A new complex, called the alternative pathway C3 convertase (C3bBb), is formed that is able to cleave C3 to form more C3b. This complex alone is unstable, but is made more stable by properdin (C3bBbP). Because the initial spontaneous cleavage of C3 leads to further cleavage of C3, the alternative pathway serves as a positive feedback loop. Additionally, the alternative pathway can serve as a way to amplify C3b coating on bacteria that may be initiated via the classical pathway.
The more recently described lectin pathway is similar to the classical pathway, but initiates activation in a different way. The first component of the lectin pathway is MBL. Like C1q, MBL is associated with proteases, MASP-1, MASP-2 and MASP-3, forming an enzyme complex that is activated when MBL binds a pathogen surface via its lectin domains. This complex is able to cleave C4 and C2, leading to complement activation and ultimately cleavage of C3.
Complement proteins C3, C5 and proteins of the membrane attack complex (C6, C7, C8 and C9) have distinct and important roles in complement function. C3b, the cleavage product of C3 that binds to the pathogen surface, is a very effective opsonin, meaning it is able to coat the pathogen surface and interact with receptors on the host cells to promote phagocytosis. C5 is downstream of C3 in all three complement activation pathways. C3b on the surface of bacteria combined with other upstream complement components is able to form the C5 convertase. This complex is able to enzymatically cleave C5. Next, C5b is added to the complex on the pathogen surface, and C5a is released. C5a is a powerful chemoattractant and inflammatory mediator, meaning it is able to recruit immune cells to the site of infection and promotes inflammation of infected tissue. After C5 is activated, the complement complex on the pathogen surface is able to recruit the membrane attack complex (also known as MAC or TCC, for terminal complement components). MAC is composed of complement proteins C6, C7, C8 and C9. These proteins are able to form a pore in bacterial membranes, leading to lysis and destruction of the bacteria.

Complement system synthesis
The liver is the primary site for complement synthesis, but complement is also expressed in other tissues. Complement proteins that are produced in the liver are secreted into the blood and circulated throughout the body. However, the body does not appear to rely solely on complement proteins from the circulation, because messenger RNA for at least some complement components has been detected in the spleen, thymus, heart, brain, intestine and kidney. The liver produces an estimated 90% of complement protein found in the serum, but the origin of complement proteins found outside the serum is more difficult to determine. Complement that is synthesized at the site of infection is hypothesized to play an important role in the immediate, tissue specific immune response.

Complement regulatory proteins (CRegs)
The complement system is finely regulated by a number of complement regulatory proteins. Complement regulatory proteins limit complement activation at different points of the complement cascade, and exist as either membrane-bound proteins or soluble proteins.

1. Membrane-bound complement regulatory proteins
Membrane-bound complement regulatory proteins primarily include complement receptor 1 (CR1), complement receptor of the immunoglobulin superfamily (CRIg), membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59.
Complement receptor 1 (CR1), also known as CD35, is expressed on the surface of erythrocytes and many nucleated cells. By accelerating the decay of C3 and C5 convertases, CR1 limits complement activation. The complement receptor of the immunoglobulin superfamily (CRIg) is found on the surface of macrophages, and another name for CRIg is VSIG4. CRIg specifically inhibits the formation of the alternative pathway C3 and C5 convertase. Membrane cofactor protein (MCP, CD46) is expressed on almost all cells except erythrocytes. As a cofactor for complement factor I, it contributes to the cleavage of C3b. Decay-accelerating factor (DAF, CD55) contains a glycosyl-phosphatidyl-inosotol (GPI) anchor and is widely expressed. DAF inhibits complement activation by facilitating the decay of the C3 convertase. Unlike the above mentioned membrane-bound complement regulatory proteins, which limit complement activation at the step of C3 and/or C5 convertase, CD59 regulates complement activation at the last step – MAC formation.
CD59 (protectin) is a small, globular protein with a molecular weight of ~20 kDa. It is expressed on almost all “self” cells, including most nucleated cells and erythrocytes. The CD59 encoding gene consists of 4 exons: exon I, II, II and IV. Exon I is not translated. Exon II encodes the hydrophobic leader sequence. Product of exon III is the N-terminal portion of the mature CD59, and exon IV encodes the C-terminal portion of the mature CD59, including the glycosylphosphatidylinositol (GPI) anchor.
CD59 inhibits the interaction between C8 and the first molecule of C9; therefore, the first C9 could not undergo conformational change and is not integrated into the cell membrane. Failed conformational change of the first C9 limits the incorporation of additional C9 molecules into the C5b-9 complex, and prevents the formation of MAC and pore formation in the cell wall. In addition to blocking MAC formation, CD59 also interferes with the ion channel formation by C5b-8 and the small-size C5b-9. Consequently, no leaky pores can be formed on CD59 expressing cells.

2. Soluble complement regulatory proteins
Major soluble complement regulatory proteins include C1 inhibitor (C1-INH), complement factor I (CFI), complement factor H (CFH), C4 binding protein (C4BP), carboxypeptidase N, vitronectin and clusterin. C1-INH inactivates C1r, C1s, and MASP-2, so that the classical and the lectin pathways are shut down in the presence of C1-INH. CFI and CFH act together to cleave C3b and accelerate the decay of the alternative pathway C3 convertase. C4BP is the cofactor for CFI during the cleavage of C4b, so it helps in facilitating the decay of the classical pathway C3 convertase. Carboxypeptidase N inactivates anaphylatoxins C3a and C5a by cleaving the Arginine within them. Both Vitronectin and clusterin inhibit the complement activation and MAC formation by binding to the lipophilic portion of C7.
Membrane bound complement regulatory proteins are also present in the soluble form. For example, CD59 is usually regarded as a membrane-bound protein since it has a GPI anchor at the C-terminal. However, it can be cleaved into a soluble form via unknown mechanisms. Thus, two forms of CD59 are normally detected: the membrane-bound form and the soluble form.

Complement system and autoimmune diseases
The intricate organization of complement is tightly controlled in the healthy body, but dysregulation of complement is often associated with damage to host tissues. Complement deficiencies resulting from genetic mutations are rare in humans, but are frequently associated with autoimmune disease. The association between complement deficiencies and systemic lupus erythematosus (SLE, an autoimmune disease that can affect several different tissues, and that is typically associated with production of antibodies against host cells) has been well characterized. The percentage of patients with C1, C4 and C2 deficiencies who develop SLE is 90%, 75% and about 15%, respectively. Interestingly, deficiency in a complement regulatory protein, that normally inhibits excess complement activation, can result in depletion of complement proteins. For example, Factor H and Factor I are complement regulatory proteins that prevent excess complement activation; individuals deficient in one of these proteins are depleted of C3. This depletion can lead to autoimmune disorders as well as an increased susceptibility to disease. Certain membrane-bound complement regulatory proteins help the complement system distinguish between self-tissues and foreign surfaces. Absence of these complement regulatory proteins can result in MAC formation on host cells and subsequent tissue damage. For example, mice made deficient in CD59, a membrane bound complement regulatory protein, have uncontrolled MAC formation and develop paroxysmal nocturnal hemoglobinuria (PNH), a condition in which erythrocytes are destroyed by MAC. In many cases total removal of a complement protein is not necessary to cause disease, as individuals heterozygous for a complement gene disruption can also have symptoms. The effects of altering the expression of just one complement protein underscore the interdependence of the complement components and the connection between levels of complement proteins and regulation of complement activity.

Complement system references
1. Sunyer J O, Lambris J D. Evolution and diversity of the complement system of poikilothermic vertebrates[J]. Immunological reviews, 1998, 166(1): 39-57.
2. The human complement system in health and disease[M]. Informa Health Care, 1998.
3. Laufer J, Katz Y, Passwell J H. Extrahepatic synthesis of complement proteins in inflammation[J]. Molecular immunology, 2001, 38(2): 221-229.
4. Colten H R, Garnier G. Regulation of complement protein gene expression[J]. The human complement system in health and disease, 1998, 217.
5. Walport M J. Complement and systemic lupus erythematosus[J]. Arthritis Res, 2002, 4(Suppl 3): S279-S293.
6. Zipfel P F, Skerka C. Complement regulators and inhibitory proteins[J]. Nature Reviews Immunology, 2009, 9(10): 729-740.
7. He J Q, Wiesmann C, van Lookeren Campagne M. A role of macrophage complement receptor CRIg in immune clearance and inflammation[J]. Molecular immunology, 2008, 45(16): 4041-4047.
8. Kimberley F C, Sivasankar B, Morgan B P. Alternative roles for CD59[J]. Molecular immunology, 2007, 44(1): 73-81.