Complement Cascade

What are the control proteins of the complement cascade?

1. C1INH 2. C4BP 3. Factor H 4. Factor I 5. DAF (decay accelerating factor) 6. MCP 7. CD59 (Protectin) 8. Factor P

What complement proteins are activating enzymes?

1. C1r 2. C1s 3. C2a 4. Bb 5. D 6. MASP-2

What is the importance of the C3b deposition? It's two-fold.

1. C3b is a component of the C5 convertase, and is needed for the eventual formation of the membrane attack complex. 2. C3b (and its breakdown products) are the opsonins that are permanently deposited on the surface of pathogens, targeting them for uptake and destruction by phagocytes.

What complement proteins are membrane-binding proteins and opsonins?

1. C4b 2. C3b

What complement proteins are peptide mediators of inflammation?

1. C5a 2. C3a 3. C4a

What complement proteins are membrane attack proteins?

1. C5b 2. C6 3. C7 4. C8 5. C9

What is C3b(2),Bb also known as? What does it do?

C5 convertase enzyme. This enzyme initiates a sequence that results in formation of the membrane attack complex.

If the C3b protein binds to a pathogen it becomes a ligand for what?

Factor B binding.

In order of most efficient in activation of the complement cascade to the least efficient how do the TG proteins rank?

IgM is the champion, followed by an impressive IgG3, then IgG1, IgG2, and both versions of IgA.

What is hereditary angioneurotic edema, or HANE? How is it treated?

Inherited deficiency of functional C1INH that results in the overproduction of anaphylatoxins, resulting is a serious illness that is characterized by systemic edema. The name of this syndrome is hereditary angioneurotic edema, or HANE, and the treatment is via replacement therapy (monthly injection of C1INH).

In an immunocompetent person, the alternative pathway always does what with regard to the other two pathways?

It amplifies them.

Describe C4BP (C4 binding protein).

It binds to the C4b component of the classical pathway C3 convertase, causing a displacement of the C2a fragment and making the C4 fragment susceptible to cleavage by factor I, permanently disabling that copy of the C3 convertase enzyme. It serves to limit the duration of the complement cascade.

What is the purpose of the membrane attack complex (MAC)?

It causes the direct killing of the pathogen by disruption of its outer envelope, forming membrane pores.

C3b is also a component of what other important cascade product?

It is a component of the C5 convertase enzyme that is required for activation of the membrane attack complex.

Which C3 convertase enzyme is activated following the activation of the lectin or classical pathway?

C4b,C2a

In what order do the complement pathways act? (consider innate vs. acquired)

1. Alternative pathway 2. Lectin pathway 3. Classical pathway (both innate and acquired)

What are the three functions of the complement cascade, irrespective of how the cascade is activated?

1. Opsonization of pathogens, making them more likely to be taken up and destroyed by a phagocyte. 2. Recruitment of inflammatory and innate cells (primarily phagocytes) 3. Direct killing of pathogens.

What are the three end products/results of the complement cascade?

1. Recruitment of inflammatory mediator cells a. anaphylatoxins 2. Opsonization of the pathogen by depositing C3b on its surface. 3. Perforation of pathogen cell membranes b. membrane attack complex (MAC)

The "turn-over" of C3 proteins results in what two protein fragments?

1. The small fragment, C3a, which is an anaphylatoxin. 2. C3b that has an exposed thioester bond that will cause it to bind covalently to the next thing that it comes into contact with, unless it is hydrolyzed first, which happens quickly.

What are the important things to know about the complement components?

1. They are produced constitutively in the liver. 2. They are secreted in their inactive zymogen form. 3. They are always available in the circulation and in extravascular fluids throughout the body. 4. Overactivation can lead to depletion of complement proteins because they are produced at steady state levels. 5. MBL is the only one not expressed at a steady state.

What immune cells bear complement receptors?

1. phagocytes 2. B cells 3. Follicular dendritic cells

Describe paroxysmal nocturnal hemoglobinuria.

A genetic deficiency.This deficiency results in the inability to produce a phosphoinositol glycolipid tail that anchors both decay accelerating factor and CD59 (or protectin) into the membrane of host cells. People that suffer from this deficiency suffer from periodic episodes of intravascular erythrocyte lysis

Describe DAF (decay accelerating factor/CD55).

A membrane-bound protein that gets its name from its ability to dissociate C3 convertase enzymes. It is a critical complement control protein. You will also need to know that DAF is also designated as CD55 (a fact that will become useful when you are learning hematology).

What type of feedback loop governs the complement cascade?

A positive feedback loop that continues to amplify itself (in the absence of regulation) until complement components are depleted.

Which C3 convertase is activated following the activation of the alternative pathway?

C3b,Bb

C3b,Bb can also be a ligand for what binding? What is the resulting product?

C3b. Product: C3b(2),Bb

What protein is involved when the classical pathway is activated in an innate capacity?

c-reactive protein

The classical pathway in the complement cascade is activated via what?

Antigens and antibodies coming into contact.

Describe bacterial pathogens and capsular material.

Bacterial pathogens that produce a capsule are almost impossible for phagocytes to recognize. The capsule encloses all other surface structures on the bacterium, and there are no phagocyte receptors that recognize capsular material.

How is the formation of C5 convertase in the classical pathway analogous to that of the alternative pathway?

Because it is formed when C3b binds to the C3 convertase of the classical pathway (C4b,C2a) to form C4b,2a,3b.

When can phagocytes recognize encapsulated bacteria?

Because phagocytes have complement receptors, they can recognize an encapsulated bacterium that has been labeled with C3b. When phagocytes recognize C3b, they phagocytize the material that C3b is bound to and destroy It.

Describe Factor H.

Binds to C3b, making it susceptible to cleavage by factor I. Once C3b is cleaved to form iC3b, it can no longer serve as a template for factor B binding, so that copy of C3b can never become part of a functional C3 convertase of the alternative pathway. Cleavage of the C3b component of the C5 convertase enzyme also deactivates that copy of C5 convertase It serves to limit the duration of the complement cascade.

What is the most important thing to know about C1?

C1 cleaves both C4 and C2, and the results of these cleavage events are the production of the C3 convertase of the classical cascade [C4b,C2a] and anaphylatoxin [C4a]). C2b is not considered an anaphylatoxin, which is probably why the designation was changed to C2b.

What complement protein binds to antigen:antibody complexes and pathogen surfaces?

C1q

What is the two complement cascade products?

C3 is cleaved to C3a and C3b.

Once C3b,Bb is formed, it becomes an active enzyme that can do what?

Cleave many copies of C3 to C3b and C3a. Note: each new copy of C3b is a new ligand for factor B binding. Therefore, this is an accelerating reaction because each of the primary products of the reaction, C3b, can be converted into an enzyme that catalyzes a reaction that produces more of that some primary product.

How are complement cascade opsonization different than Ab-mediated opsonization?

Complement opsonization is permanent as the complement proteins are covalently bound to the surface of the pathogen.

Describe a factor I deficiency.

Deficiency of factor I allows extended C3 convertase activity and for more production of C3 convertase of the alternative and classical pathways. Ultimately, this leads to depletion of C3, preventing activation of the complement cascades when they are really needed. Additionally, factor I deficiency prevents the formation of an important opsonin of complement, referred to as iC3b.

Describe the innate mechanism for activation of the classical pathway.

During septic bacterial infections, a protein known as C-reactive protein is produced in the liver and released into the circulation. CRP binds to phosphocholine residues that are found on the surface of bacteria (but not host cells). When CRP is bound to phosphocholine, it mimics an IgM molecule and becomes a ligand for C1 binding. When C1 binds, the classical pathway becomes active.

Describe the composition of a C1 molecule.

Each C1 molecule is composed of six identical C1q "stalks" and 1 copy each of the serine proteases C1r and C1s. When a copy of C1 binds to antigen-engaged antibody(s), the C1r protein becomes enzymatically active and cleaves C1s. Once C1s is cleaved, it becomes enzymatically active and is able to cleave C2 and C4 proteins, initiating the classical complement cascade.

Why is IgM so efficient in activation of the complement cascade?

Each secreted copy of IgM is a pentameric structure. When it binds to a multivalent antigen, the C1 binding site becomes available on multiple copies of the IgM Fc regions. This allows for the C1q stalks to bind to several of these Fc ligands simultaneously, causing a conformational change to C1 that initiates cleavage of C1r. In contrast, more than one copy of the monomeric antibody isotypes is required for activation of C1.

Describe CD59 (protectin)'s purpose.

It is a protein that is found on the surface of host cells. This protein prevents membrane attack complex formation on host cells by binding to the C5b,6,7,8 complex and preventing C9 from binding to the complex. This protects host cells from MAC-mediated damage. There may be some extracellular pathogens that have adapted by producing a homolog of CD59 (I suspect that some have), but most pathogens have no way to prevent MAC formation.

Why is C3b the most important product of the complement cascade?

It is the primary opsonin created by these pathways.

Describe factor I.

It is the protease that cleaves C4b once C2 has been dissociated from it by C4-binding protein, and it cleaves C3b that has been bound by factor H. Deficiency of factor I allows extended C3 convertase activity and for more production of C3 convertase of the alternative and classical pathways. Ultimately, this leads to depletion of C3, preventing activation of the complement cascades when they are really needed. Additionally, factor I deficiency prevents the formation of an important opsonin of complement, referred to as iC3b.

Describe the passive regulation of the complement cascade.

It is the result of the rapid hydrolysis of the thioester bonds that are made available on the large fragments of C2, C3, C4, C5, and Bb following their cleavage. They must bind to something before they are hydrolyzed, or they become inert.

Once factor B has bound to C3b, what happens?

It undergoes a conformational change that makes it susceptible to cleavage by factor D.

How does the C5 convertase enzyme work?

It will cleave any C5 proteins it engages with, resulting in a large fragment (C5b, the 1st component of the MAC) and a small fragment (C5a) that is a potent anaphylatoxin. In fact, C5a is a very important chemotactic factor for neutrophils.

The lectin pathway in the complement cascade is activated by what?

Lectin binding to pathogen surfaces.

What complement protein binds to mannose on bacteria?

MBL

How does the lectin pathway work and what other pathway does it mimic in the end?

MBP binds to exposed mannose residues on the surface of bacteria. The MBP undergoes conformational changes that result in it becoming a ligand for binding by mannan-binding lectin-associated serum proteases, or MASP proteins (MASP-1 and MASP-2). Once MASP proteins bind to MBP, they become enzymatically active and cleave copies of complement components C2 (to create a large fragment called C2a and a small fragment called C2b) as well as C4 (to create a large fragment named C4b and a small fragment called C4a). When C4b becomes bound by C2a, the product is the C3 convertase of the classical pathway (C4b,C2a). From this point forward, the lectin pathway is identical to the classical pathway.

In the lectin pathway, what binds to the mannose residues on bacteria?

Mannose-binding protein (MBP)

Describe MCP (membrane cofactor protein).

Membrane-bound protein on host cells. When MCP binds to either C3b or C4b, they become susceptible to cleavage by factor I. Therefore, this protein is important for shutting down C3 convertase activity.

Describe CR1.

Membrane-bound protein that binds to C3b and C4b, making them susceptible to factor I cleavage.

Once C5b is generated by the C5 convertase enzyme, how does the MAC come to be?

Once C5b has been generated, it binds to one copy of C6 and one copy of C7 to form the C5b,6,7 complex. This complex then inserts into membranes (outer envelope of bacteria, for instance). Once embedded in the membrane, C5b,6,7 becomes a ligand for C8 binding, and then 10-16 molecules of C9 bind to that complex forming a pore in the membrane.

The alternative pathway in the complement cascade is activated by what?

Pathogen surfaces.

How do RBCs facilitate immune complex removal?

Small immune complexes, which consist of antigen that is bound by either antibodies, C3b, or both are not efficiently taken up by phagocytes. If they are allowed to build up in the circulation, they can cause vasculitis and kidney dysfunction (we will discuss at length later). One of the important roles of erythrocytes is the transport of small immune complexes to the liver and spleen (blood filters that have many resident macrophages). Macrophages in these tissues remove these immune complexes from the RBCs and destroy them.

Describe the specificity functions, and cell types that possess C3a receptors.

Specificity: C3a Functions: Binding of C5a activates G protein Cell types: Endothelial cells, mast cells, and phagocytes.

Describe the specificity, functions, and cell types that possess CR1 receptors.

Specificity: C3b, C4bi, C3b Functions: Promotes C3b and C4b decay, stimulates phagocytosis, erythrocyte transport of immune complexes Cell types: Erythrocytes, macrophages, monocytes, polymorphonuclear leukocytes, B cells, and FDC.

Describe the specificity functions, and cell types that possess CR2 receptors.

Specificity: C3d, iC3b, C3dg, Epstein-Barr virus Functions: Part of B-cell co-receptor, Epstein-Barr virus receptor. Cell types: B cells, and FDC.

Describe the specificity functions, and cell types that possess C5a receptors.

Specificity: C5a Functions: Binding of C5a activates G protein Cell types: Endothelial cells, mast cells, and phagocytes.

Describe the specificity functions, and cell types that possess CR3 (Mac-1, CD11c/CD18) receptors.

Specificity: iC3b Functions: Stimulates phagocytosis Cell types: Macrophages, monocytes, polymorphonuclear leukocytes, and FDC.

Describe the specificity functions, and cell types that possess CR4 (gp150,95, CD11c/CD18) receptors.

Specificity: iC3b Functions: Stimulates phagocytosis Cell types: Macrophages, monocytes, polymorphonuclear leukocytes, and dendritic cells.

Describe the active regulation of the complement cascade.

The active regulation depends on the activity of several regulatory proteins, some of which are soluble, while the others are embedded on the surface of host cells.

Which two complement pathways are purely innate?

The alternative and lectin pathways

Which complement pathway is considered to be an acquired immune response?

The classical pathway

The cleavage of factor of C3b by factor D results in what?

The formation of C3b,Bb, or C3 convertase of the alternative pathway. Note: if C3b binds to a host cell, complement control proteins prevent the activation of the complement pathway.

Neither the lectin or classical pathways are able to amplify the alternative pathway because of why?

There is no product of the alternative pathway that can initiate either the lectin or classical complement pathways.

What are the purpose of opsonins?

They are easily recognized by phagocytes, encouraging them to engulf and destroy the pathogens.

What is the purpose of anaphylatoxins?

They are inflammatory mediators that activate vascular endothelium and recruit phagocytes to the inflammatory site. They are chemotactic and induce vascular permeability

Describe the complement receptors of phagocytes.

They express an array of complement receptors that allow them to recognize materials that have been opsonized with complement component C3b (or its various breakdown products).

Describe the Fc receptors for phagocytes.

They have Fc receptors for IgGs and IgAs (there are no Fc receptors for IgM...important to remember). Antibodies also serve as opsonins because if a phagocyte recognizes antibody-"labeled" material, it phagocytizes the material and destroys it.

How is the Classical pathway initiated?

This pathway is initiated when complement component C1 binds to antibody that is bound to its cognate antigen (like a bacterial surface structure). C1 has affinity for a region of the Fc portion of some antibody isotypes that becomes available for binding when the antibody is bound to its cognate determinant. Or (another way of explaining it) This pathway is initiated when complement component C1 binds to the Fc region of an antibody that is bound to its specific antigen. C1q is responsible for this binding.

Describe the C1 inhibitor.

This regulatory protein binds to activated C1r:C1s, forcing them to dissociate from C1q. This halts the cleavage of C2 and C4 proteins by that particular copy of C1, thus dampening the complement cascade. C1 inhibitor is particularly important for controlling activation of C1 that continuously occurs spontaneously at a low level in the serum and extravascular fluids. As you will see in a few minutes, C1 inhibitor is a very important molecule from a clinical perspective.

How many versions of the C3 convertase enzyme are there?

Two 1. C3b,Bb 2. C4b,C2a

How is the complement cascade regulated?

Via passive and active regulation.