The complement system Flashcards
What is the complement system?
The complement system is a group of >50 serum proteins and glycoproteins that are circulating in it’s inactive form (zymogens) that cooperates with both the innate and the adaptive immune systems to eliminate pathogens, dying cells, and immune complexes from the body through many different functions, and we still don’t know all of it.
The proteins in the complement system can be divided into seven broad categories based on their function, which?
- Initiators: proteins that bind to ligands, either directly or via an antibody or other pathogen-specific protein and induce a conformational change which leads to altered activity.
- Enzymatic mediators and convertase activators: proteolytic enzymes that cleave and activate the next member of a complement reaction sequence, to generate the central proteins of the complement cascade, eg the C3 and C5 convertases, which cleave C3 and C5, releasing active components that mediate functions of complement (like 3, 4, 5)
- Opsonins: Effector complement proteins, fragments formed after proteolytic cleavage that serve as opsonins - ligands that bind covalently to microbes/pathogens which “tag” it for phagocytosis through serving as ligand for receptors present on phagocytic cells, eg C1q, C3b.
- Anaphylotoxins: Inflammatory mediators - Some small complement fragments act as inflammatory mediators through binding to receptors on the endothelial cells lining small blood vessels and induce an increase in capillary diameter, thus enhancing blood flow to the affected area. They also attract other cells to the site of tissue damage.
- Membrane attack complex (MAC): Proteins that form pores in the membrane of pathogens resulting in the lysis of the pathogen. MACs can also form on infected host cells, although the complement system must first overcome the regulatory mechanisms designed to protect host cells from complement attack.
- Complement receptors: complement receptors on phagocytic cells, granulocytes, or erythrocytes that when bound to complement activators induce the effector functions: Trigger phagocytosis (opsonins) or trigger degranulation in neutrophils.
- Complement regulators: Regulatory proteins limit the effects of complement by promoting their degradation or preventing their binding to host cells to stop the cascade. For example factor I, which degrades C3b, and CD59 (protectin), which inhibits the formation of the MAC on host cells.
Where are the complement proteins synthesised?
Mostly in the liver by hepatocytes, but are also produced by blood monocytes, tissue macrophages, fibroblasts, and epithelial cells of the gastrointestinal and genitourinary tracts.
What is meant by the term “complement cascade”?
The sequence of proteins in a complement pathway from the initiator protein to the biological effector is referred to as a “complement cascade.”
There are three pathways for initiation of the complement cascade, which and how do they differ mainly?
The classical pathway, the lectin pathway and the alternative pathway. All pathways consist of three key steps, initation, amplification and termination. The route of initiation is different and converge in the amplification step for the classical and lectin pathway, but in the end - all pathways result in the generation of an enzyme complex that cleaves the C3 molecule, C3 convertases that result in a major increase of C3b - a key complement protein.
One of the complement activation pathways need components of the adaptive immune system, which?
The classical pathway of complement activation have a strong connection to the adaptive immunity, because antibodies bound to antigen, also referred to as immune complexes are needed to activate the pathway, specifically antibodies of the IgM class or some subclasses of IgG.
Note, IgM is more effective in activating complement as it is a pentamer.
Describe the classical pathway for complement activation in detail.
- An antibody-antigen complex is formed, and upon this binding a conformational change occurs in the Fc region of the antibody, exposing the three binding sites (only two needed) for the complement protein C1q in the C1qr2s2 complement complex. When C1q has bound, it activates one of the C1r subunits by changing its conformation into a protease, which in turn activates the other C1r and they go on to cleave the the two C1s molecules which activates them.
- The activated C1s molecules cleaves off a fragment of the C4 N-terminus (on one chain) through hydrolysis, C4a. The remaining C4 fragment, C4b binds covalently to the target membrane. C4b binds to C2 and causes a conformational change that exposes C2 to the action of C1s which hydrolyse it, cleave off C2b and forms C4b2a, the C3 convertase.
- The C3 convertase go on to cleave/hydrolyse many C3 molecules, forming the small anaphylotoxin fragment C3a and the extremely important complement component C3b, that is an essential precursor to many downstream complement reactions. A single C3 convertase molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step of the classical pathway.
- The C3b component of the C5 convertase binds C5 and cleave it into the anaphylotoxin C5a and C5b, a component in the membrane attack complex (MAC).
The C3b complement protein is essential and have three important roles, which?
The three functions of the C3b complement protein is:
- it can bind to microbial surfaces as an opsonin, tagging them for opsonization - complement facilitated phagocytosis.
- It can bind to the Fc part of antibody-antigen complexes, tagging them for phagocytosis or destruction in the liver.
- It can bind the membrane bound C4b2a complex, forming the C5 convertase, C4b2a3b.
In the classical pathway of complement activation, how is binding of complement to non-antigen bound IgM hindered?
For a stable C1q-antibody interaction to occur, each C1 macromolecular complex must bind to at least two antibody constant regions. When circulating unbound, the IgM pentamer have a planar conformation that hides the C1q binding sites, but when it binds to an antigen with more than one monomer (multivalent antigen), the whole pentamer changes conformation to become more rounded, like a shield - exposing the binding sites of the C1q complement protein and facilitating a stable C1q-antibody interaction. This ensures that only an antigen bound antibody can initiate the classical pathway of activation of complement.
Monomeric IgG only have one binding site for C1q with low affinity, so polymerisation of IgG is needed to create a stable interaction and initiate complement. The more IgG in the complexes, the stonger the binding, allowing for fine tuning. This also ensures that complement is only activated (through the classical pathway) when IgG is antigen bound.
Give one example of how the classical pathway is regulated.
The C4 fragment C4b binds covalently to the target membrane through a highly unstable thioester (C-S) reacting with an amino group on the membrane or on the FC part of the antibody-antigen complex, but this must happen quickly before the thioester is hydrolysed (90% of C4b are hydrolysed before binding (C-OH)). If no target is close, the bond is hydrolysed. Also, having this unstable binding ensures that not too much binds and forms C3 convertase complex, as one of these complexes can generate up to 200 C3b molecules, which are potent.
Explain the classical activation of complement in short.
The classical pathway is initiated by antibodies of the IgM or IgG class binding to a multivalent antigen. Next, C1 binds to an antibody, activating C1-associated serine proteases that cleave the second and fourth complement components, releasing C2a and C2b and C4a and C4b fragments. C2a and C4b combine to form an active serine protease, called C3 convertase, that cleaves C3 into C3a and C3b. The C2a4b complex then combines with one molecule of C3b, forming an active serine protease enzyme called C5 convertase that cleaves C5 into C5a and C5b.
How is the lectin pathway of complement activation different from the classical pathway?
The lectin pathway is initiated by lectins - proteins that recognize particular carbohydrate components- as receptor molecules instead of antibodies and is thus considered to be an arm of the innate immunity. It is only different in the initiation compared to the classical pathway.
Give an example of a lectin capable of activating complement and describe how and which antigens it recognizes.
One example of a lectin capable of activating complement is mannose-binding lectins (MBL), that can bind sugars such as mannose, N-acetylglucosamine, D- glucose, and L-fucose polymers. Mannose is present on the surfaces of microbes such as Salmonella, Listeria, and Neisseria bacteria; Cryptococcus neoformans and Candida albicans fungi; and even on the membranes of some viruses such as HIV- 1 and respiratory syncytial virus (RSV). MBL recognizes their associated hydroxyl groups in defined three-dimensional arrays, making it function as a classic PRR.
How is the lectin pathway of complement activation initiated?
In the blood, MBL is associated with MBL-associated serine proteases (MASP proteins), mostly MASP-2 which is structurally similar to C1s (classical pathway). When MBL binds antigens in the blood, MASP-2 cleaves C4 and C2 in the same way as in the classical pathway, giving rise to the C4b2a C3 convertase. Downstream of that, it has the same pathway as the classical pathway.
The alternative pathway of complement differs quite a lot from the others. Compare the alternative pathway to the lectin and classical pathway in terms of defferences and similarities.
All the pathways converge in the formation of the membrane attack compex eventually leading to lysis of the microbes. The alternative pathway is similar to the lectin pathway in that is does not rely on antibodies and thus is considered to be part of the innate immunity. The differences are more prominent:
- The alternative pathway can be activated in three different ways
- it uses a different set of C3 and C5 convertases, containing the Bb component from factor B.
Describe one of the three alternative pathways to complement activation in short.
The alt “tickover” pathway:
The complement component C3 is contitutively expressed and is always present in the serum in high concentration. The tickover pathway is initiated when its internal thioester bond is spontaneously hydrolyzed, C3(H2O). In the presennce of mg2+, the C3(H2O) can bind factor B and the complex is susceptible to cleavage by the protease factor D, which cleaves factor B into a small Ba subunit and a catalytically active Bb subunit still bound to C3(H2O) and forms the C3(H2O)Bb complex, also referred to as the “fluid-phase C3 convertase” because it remains in the blood plasma and is not bound to any cells.
The fluid-phase C3 convertase goes on to hydrolyze many C3 moolecules into C3a and C3b. The C3(H2O)Bb complex is not very stable and is degraded rapidly in healthy hosts, but when an infection is present, the newly formed C3b molecules can bind to the surface of nearby microbes. Once C3b have bound to the microbe surface, Factor B can bind and can be cleaved by Factor D to form C3bBb complexes. These C3bBb complexes are now located on the microbial membrane surface and have C3 convertase activity, although it needs too be stabilized by properdin (factor P). The stabilized membrane bound C3 convertase go on to amplify the number of C3b with further factor B and D involvement. C3b can then bind to the C3bBb C3 convertase complex to form the alternative C5 convertase complex, with the composition C3bBbC3b (also stabilized by properdin) that can go on to cleave C5 to C5b - the initiatior of the MAC.
Note that the inital step of this pathway is spontaneous and happens without antigen presence. Eva is sceptical of wether this occurs under physiological conditions as it would be dangerous to spontaneously activate complement, so take it with a grain of salt.
The other patways are: The alt Properdin-Activated Pathway and the alt Protease-Activated Pathway.
All the complement activation pathways culminate in the formation of C5b. What is the function of this complement protein?
C5b is the initiator of the membrane attack complex (MAC). However, the C5b component is extremely labile and is not covalently bound to the membrane, as are C3b and C4b. Therefore, it is rapidly inactivated unless it is stabilized by the binding of the serum protein C6.
Explain the formation of the membrane attack complex.
- C5b binds C6 and is stabilized - the complex interacts reversibly with the membrane.
- C7 binds to the C5bC6 complex and the binding induces a conformational change in the C7 protein exposing a hydrophobic region capable of insertion into the microbial membrane. Insertment is the triggering event for the formation of the membrane attack complex.
- C8 is made up of two peptide chains: C8β and C8αγ. Binding of C8β to the C5b67 complex induce a conformational change in the C8αγ dimer to expose hydrophobic regoins that also inserts into the membrane, creating a small pore.
- Binding of and polymerization of C9 to the C5b678 complex forms the MAC that causes loss of plasma membrane integrity and untimately lysis of the cell. During polymerization, all the C9 molecules undergo conformational changes so that they too can insert into the membrane. As many as 10 to 19 molecules of C9 can be bound and polymerized by a single C5b678 complex.
How is MAC formation regulated?
The triggering event of the formation of MAC is when C7 inserts into the membrane, and if the binding of C7 to C5bC6 occurs on a surface that is not a membrane, like on an immune complex or other non-cellular surface, the complex will be released. This ensures that the MAC is only formed when it should.
Which are the three direct defense functions of the complement system? Which complement proteins are involved?
- Lysis of bacterial and cell membranes: Membrane attack complex (C5b-C9).
- Opsonization: Covalently bound C3b, C4b, C1q.
- Induction of inflammation and chemotaxis by anaphylatoxins: C3a and C5a (anaphylatoxins) and their receptors on leukocytes.
The complement receptors are a diverse group with many functions, give two examples.
Ex 1: CR2 (complement receptor 2 - aka CD21) a B cell coreceptor that binds to breakdown products of C3b (like C3d) on antigens, the additional binding of the antigen via C3d provides a higher affinity binding of the antigen, and works as costimulatiory signal to B cells, enhancing B-cell activation.
Ex 2: C3aR/C5aR (GPCRs) mainly on granulocytes bind the anaphylotoxins C3a and C5a as ligands which stimulates degranulation and release of pro-inflammatory cytokines.
Ex 3: CR1 (CD35) present on leukocytes and erythrocytes, and have very diverse effector function depending on the cell type it’s expessed on. The CR1 binds with high affinity to C3b and smaller C3b breakdown products, C4b, C1q, and MBL as ligands. Erythrocytes bind immune complexes via their CR1 receptors and transport the complexes to the liver, where they are picked up by phagocytes and cleared from the body. CR1 on B cells mediates uptake of C3b-bound antigen, leading to its degradation in the B-cell lysosomal system and subsequent presentation of antigenic peptides to T cells. It’s both expressed by the innate and adaptive cells.
What are the three main effector activities of the complement system?
- Direct defence via MAC, opsonins and inflammation
- linking the innate and adaptive defence together, mostly to enhance.
- involvement in the contraction phase of the immune response: clearance of immune complezes and apoptotic cells and induction of regulatory T cells, all to stop the immune response.
Page 397.
The complement system provides an interface between innate and adaptive immunity, how?
- Some complement proteins function as co-receptors on B cells (CD21) and thus function through augmentation of antibody responses
- Enhancement of antigen presentation: DCs and macrophages express all known complement receptors.Effects on: Antigen uptake, Migration, Interleukin production
- Induction of regulatory T cells: Complement participates in quality control mechanisms during T
cell development and supports their growth, differentiation and
survival. - Complement proteins are involved in the enhancement of immunologic memory
The complement system is both regulated passively and actively, which mechanisms provide passive regulation?
The passive regulation of the complement system consists of:
- Short half lives of many key complement proteins: This protects against extended periods of inadvertent/unintended complement activation.
- Different composition of extracellullular components on host cells vs microbes: Many host cells have high affinity to negative regulators of complement, which ensures that any complement proteins ending up on host cells are destroyed rapidly.
There are five possible mechanisms by which the complement system can be actively regulated, which?
All the active regulatory mechanisms are dependent on several regulatory
proteins that perform a regulatory action:
- dissasociation: eg of the C1 components
- decay acceleration: eg of the C3/C5 convertases.
- degradation: eg by factor I that degrades C3b and C4b
- inhibition of lysis: eg by CD59 (Protectin) that inhibits polymerization of C9, thus inhibiting MAC formation
- cleavage: anaphylatoxin activity is regulated by cleavage of the C-terminal arginine residues from both C3a and C5a by serum carboxypeptidases, resulting in rapid inactivation of their anaphylatoxin activity.
Give two examples of active regulatory mechanisms of the complement system.
Ex 1 Dissassociation:
The C1 inhibitor C1INH functions as an serine protease inhibitor by binding to their active site. C1INH functions by binding and forming a complex with C1r2s2, causing it to dissassociate from C1q - thus hindering the first step of the classical pathway, cleavage of C2 and C4. It also promotes dissassociation of MASP-2 from mannose-binding lectins (MBL) so it can inhibit the lectin pathway of complement activation too.
Ex 2 Decay acceleration:
The membrane-bound decay-accelerating factor, or DAF (CD55), accelerates the decay of the C4b2a C3 convertase (resposible for a major amplification step) on the surface of host cells. In order to complete its job, DAF requires the cofactors CR1 and C4BP (C4-binding protein). ¨
Ex 3 Degradation:
Factor I is a soluble, constitutively (always) active serine protease that can cleave membrane-associated C3b and C4b into inactive fragments. But it requires the membrane-bound host cell proteins C4BP, and CR1 as cofactors (and Factor H) which makes it only work on host cells.
Ex 4: CD59 (protectin), binds any C5b678 complexes that may be deposited on host cells and prevents their insertion into the host cell membrane through blocking further C9 addition to developing MACs. In addition, the soluble complement S protein, otherwise known as vitronectin, binds any fluid-phase C5b67 complexes released from microbial cells, preventing their insertion into host cell membranes.
What would be the outcome of haviing homozygous deficiencies in any of the early components of
the classical pathway?
Having deficiencies in the early stages of the classical pathway (C1 cleaving C4 and C2 and in turn forming C3 convertase which converts C3 into C3b) you would have limited protection against pathogens without the sugars on their surface that can be recognized by the lectin pathway. Also, since C1q is involved in clearance of apoptotic cells so you coould develp autimmune disorders like SLE.
What would be the outcome of having MBL-deficiency?
Since MBL is a lectin pathway initiator deficiencies can lead to recurrent infections by pathogens with good evasion strategies to the other complement pathways.
–> increased serious fever-inducing infections in children
What would be the outcome of having C3 deficiencies?
C3-deficiencies are behind the most severe clinical manifestations of
complement deficiencies.
Since C3 is involved in all the complement pathways, there woudl be no C3b which means no opsonization, no immune complex clearance and no downstream MAC formation. You would basically not have any complement protection to infection, but you would still be able to clear apoptotic cells thanks to having C1 intact.
–> frequent bacterial infections and immune-complex disorders
Provide an example of a disease you could have if you have deficiencies in regulatory complement proteins and what the outcome is.
An example of a disease you can get with deficiencies in regulatory complement proteins is heredetary angioedema, where you have deficiencies in C1INH - which regulates activation of the classical pathway by preventing excessive C4 and C2 activation by C1. However, as a serine protease inhibitor, it also controls two serine proteases in the blood clotting system. Patients with C1INH deficiency suffer from a complex disorder that includes excessive production of vasoactive mediators (molecules that control blood vessel diameter and integrity), which in turn leads to tissue swelling and extracellular fluid accumulation.
Give three examples of complement evasion strategies by microobes.
- Some mechanisms interfere with the first step of classical complement activation by binding to the Fc regions of antibodies (e.g. staphylococcal protein A)
- Microbial proteins may bind and inactivate complement proteins, masking their sites of interaction (e.g. S. aureus proteins)
- Microbial proteases destroy complement proteins (e.g. Pseudomonas & Streptococci)
- Some microbes mimic or bind complement regulatory proteins (e.g.
Streptococcus pyogenes, Variola and vaccinia viruses). - Some also induce the synthesis of regulatory proteins by the cells that harbor them
Order the three coomplement activation pathways from oldest to newest.
Alternative is the oldest, lectin pathway next and newest is the classical pathway as it requres the adaptive immune system which only vertebrates have.
