5 - The Complement System Flashcards
General overview
Classic:
1) IgG/IgM binds pathogen
2) IgG/IgM binds C1
3) C1 splits C4 into C4a and C4b
4) C4b binds C2a (makes C4b2a aka C3 convertase)
5) C4b2a splits C3 to C3 + C3b
6) C4b2a binds C3b -> C4b2a3b (aka C5 convertase)
7) C4b2a3b splits C5 to C5a +C5b
Lectin:
1) MBL (mannose binding lectin) binds mannose on pathogen
2) MBL binds MASP
3) MBL-MASP splits C4 to C4a and C4b
4) C4b binds C2a -> C4b2a (C3 convertase)
5) C4b2a splits C3 to C3a and C3b
6) C4b2a binds C3b -> C4b2a3b (C5 convertase)
7) C4b2a3b splits C5 into C5a + C5b
Alternative:
1) C3 splits spontaneously to C3a+C3b
2) C3b binds pathogen
3) C3b binds Factor B -> C3bB
4) Factor D splits Factor B -> C3bBb (C3 convertase)
5) C3bBb splits C3 to C3a and C3b
6) C3bBb binds C3b -> C3bBb3b (C5 convertase)
7) C3bBb3b splits C5 to C5a and C5b
Common endpoint:
1) C5b binds C6-C9 -> MAC
2) MAC creates hole in the pathogen
3) pathogen dies
The seven functional categories of the complement system
1) Initiator complement components. These proteins initiate their respective complement reactions by binding to particular soluble or membrane-bound molecules. Once activated by their ligand, they undergo conformational alterations resulting in changes in their biological activity
2) Enzymatic mediators. Several complement components are proteolytic enzymes that cleave and activate the next member of a complement reaction seq. Proteins that are inactive until cleaved by proteases = zymogens. Some complement proteases become active by binding to other macromolecules and undergoing a conformational change; others are zymogens themselves, inactive until cleaved.
3) phagocytosis-enhancing components, aka opsonins. Upon activation from the complement cascade, several complement proteins are cleaved into two fragments, each taking on a particular role. C3b and C4b are examples of opsonins. they bind covalently to microbial cells and serve as ligands for phagocytes with receptors for them.
4) Inflammatory mediators. These fragments bind to receptors on the endothelial cells lining capillary, and results in vasodilation (enhancing blood flow to the area). they can also attract other cells to the area. In excess, these are harmful. The fragments are therefore called anaphylatoxins. Examples: C3a and C5a.
5) Membrane attack proteins. proteins of the membrane attach complex (MAC) insert into the cell membrane and punch holes, leading to lysis of the pathogen. MACs can also form on infected host cells, although the complement system will then have to overcome the protection host cells have.
6) Complement receptor proteins. Receptor molecules on the cell surfaces bind complement proteins and signal specific cell functions. the compl. receptor CR1 binds to C3b that have opsonized a pathogen, triggering phagocytosis.
7) regulatory complement components. Protect host cells from complement system. include factor I (degrades C3b) and CD59 (inhibits the formation of MAC on host cells)
C5 convertase
5Ca = inflammatory mediator/anaphylatoxin
5Cb = initiating factor for the formation of MACs
The classical pathway initiation
considered part of the adaptive IS
begins with formation of Ag-Ab compelxes. Can be soluble (= immune complexes), or be formed when an Ab binds to epitopes on cell membranes.
IgM / IgG (certain subclasses)
Initial activation: Ab-Ag complexes interact with C1, C2, C4 (normally found in plasma as inactive precursors or zymogens)
The Ab undergoes conformational changes, exposing binding site for C1 (C1q, 2C1r, 2 C1s). Each C1 complex needs to bind 2+ Abs for a stabel Ig/Ab interaction.
IgM molecules engaged in an Ag-Ab complex has undergone conformational changes to allow binding of C1q (3 bs).
IgG has one bs for C1q. the affinity is low before interactions with an Ag.
C1q can also bind ligands independent of IgG and IgM, also activating the complement cascade.
Classical pathway of complement activation
Main concept:
C1q binds Ag/Ab compex. Conformational change, activating the other 4 subunits.
C4 and C2 is cleaved, go together to make C4bC2a, aka C3 convertase. One C3 convertase can cleave multiple C3 molecules. Some C3b molecules will join C4bC2a, making C4bC2aC3b (C5 convertase).
C5 is split into C5a and C5b. C5a is an inflammatory cytokine. C5b can make MACs.
1) C1q binds Ag-Ab and induces a conformational change in one C1r molecule, activating it. This C1r thena ctivates the second C1r and the 2 C1s molecules.
2) C1s cleaves C4 and C2. C4 is cleaved first. C4b binds to the membrane close to C1 and binds C2m exposing it to the action of C1s. C1s cleaves C3, creating C3 convertase (C4bC2a)
3) C3 convertase hydrolyzes many C3 molecules. Some cleaved molecules combine with the C3 convertase to make C5 convertase
4) C3b component of C5 convertase binds C5, permitting C4b2a to cleave C5.
C4b has an unstable thioester bond that can reactc with OH or amino groups on proteins/carbs in the cell membrane. Approx 90% of C4b is further hydrolyzed before it can bind the cell surface due to the instability of this bond.
C3b
3 things:
1) OPSONIZATION: binds covalently to microbial surfaces (similarly to C4b), thus providing a “tag” that allows phagocytes with C3b receptors to engulf the tagged microbes.
2) Like C4b, C3b can attach to the Fc portion of Ab in soluble Ag/Ab complexes. these C3b-tagged complexes are bound by C3b receptors on phagocytes or red blood cells, and are either phagocytosed or conveyed to the liver to be destroyed!
3) Some C3b molecules bind the membrane-enzyme C4b2a (C3 convertase), and the new complex is called C5 convertase.
Lectin pathway
initiated when soluble proteins recognize microbial Ags
Innate
lectins = proteisn that rec particular carb components = receptors in this pathway (rather than Abs)
Alspo proceeds through the activation of a C3 convertase
Mannose-binding lectin (MBL) binds mannose, D-glucose, N-acetylglucosamine, and L-fucose from bacterial surfaces via their OH groups.
MASP (MBL-associated serine proteases) (1,2,3). MASP-2 = most important, as it cleaves C4 and C2.
Same results as classical pathway.
Alternative pathway
Innate. Uses different set of C3 and C5 convertases (C3bBb and C3bBbC3b, respectively). Bb is unique to this pathway.
can be initiated in 3 ways:
1) “tickover pathway”. Uses the four serum components C3, factor B, factor D and factor P/properdin.
2) initiated by properdin
3) initiated by proteases like thrombin and kallikrein
The tickover pathway
C3 is constantly made and inactivated.
C3 undergoes spontaneous hydrolysis at its internal thioester bond, yielding C3(H2O). In the presence of Mg2+, C3(H2O) binds factor B. factor B is then cleaved by factor D, into Ba and Bb. Bb is catalytically active, and remains bound to C3(H2O) as C3(H2O)Bb (aka “fluid-phase C3 convertase”). It remains in th eblood plasma and is not bound to any cells. This enzyme cleaves many C3, but is not very stable in healthy hosts and rapidly degraded. However, if there is infection, the C3b molecules bind nearby microbial surfaces.
factor B can bind C3b, and does so under infection. It then becomes susceptible to cleavage by factor D, generating C3bBb complexes on the microbial cell membrane. these have C3 convertase activity, and takes over from the fluid-phase C3 as the dominant C3 convertase. This cell-bound C3 convertase is unstable until binding of preperdin (factor P).
All C3b molecules can bind to microbial surfaces and initiate this part of the alternative pathway, regardless of which pathway the C3b was originally made from.
The C5 convertase complex is the result of the C3 convertase + C3b, and will in this case be C3bBbC3b.
The properdin-activated pathway
in addition to stabilizing the alternative pathway, properdin can also initiate it.
In lab: upon binding of properdin to an artificial surface, the rest of the cascade could be activated. This pathway relies in pre-existing low levels of C3b, which must be gererated by mechanisms such as the tickover pathway
The protease-activated pathway
Similar in concept to the blood coagulation pathway. Both use protease cleavage and conformational alterations of key proteins to modify enzyme activity, as well as amplification of various steps of the pathways by fed-forwards loops.
thrombin (involved in blood clotting) can cleave C3 and C5 in vitro, but needs relatively high [thrombin]. C5a = anaphylatoxin (proinflammatory, its generation leads to amplification of the inflammatory state). However, functionally meaningful [C5b] are not generated this way.
The three complement pathways converge at the formation of C5 convertase and generation of MAC
The C5 convertases have different compositions;
classical and lectin pathway = C4b2a3b
alternative pathway = C3bBbC3b
However, the function is the same; cleaves C5 to C5a and C5b.
C5b is generated on microbial surface, providing a bs for the rest of the components of the MAC. However, this binding is not covalent, and C5b must be stabilized by C6 to not be inactivated.
MAC actually penetrates the cell membrane. C5bC6 interacts reversibly with the cell membrane. Binding of C7 causes conformational changes in C7, exposing hydrophobic regions capable of inserting it into the membrane. this is the triggering step for the formation of MAC. C8 beta kan bind to the C5b67 complex, and induce a conformational change in the other C8 peptide chain (C8-alpha-gamma), to the hydrophobic domain of C8a-y can insert itself into the cell membrane. The C5b678 complex can make a small pore in the membrane. The final step in MAC formation is the binding and polymerization of C9 to the C5b678 complex. 10-19 molecules of C9 per C5b678. C9 undergoes conformation change so it can insert itself into the membrane (in a circle to make a large pore).
Can also be made in soluble forms if C6 and C7 binding occurs on an Ag-Ab complex. No anchoring to the complex, and is released. Could harm innocent cells, but are usually destroyed by protein S.
The diverse funcitons of complement
Innate defense (lysis, opsonization, induction of inflammation)
Interface between innate and adaptive (augmentation of Ab-responses, enhancement of immunogenic memory and Ag-presentation, potential effect on T cells)
Contraction phase of the IS (clearance of immune complexes from tissues, and of apoptotic cells, + induction of regulatory T cells).
Complement receptors connect complement-tagged pathogens to effector cells
CR1 is expressed on both leukocytes and erythrocytes. Binds C3b with high affinity. Also binds C3b breakdown products (C4b, C1q, MBL).
erythrocytes bind immune complexes via CR1 and transport them to the liver (phagocytosed)
Phagocytes and opsonization. phagocytosis + release of proinflammatory proteins
B cells: mediates C3b-bound Ag uptake, leading to degradation in B-cell lysosomal system and subsequent presentation to T cells. Also important for transport of Ag into the follicles of lymph nodes and spleen, where they are transferred to macrophager or follicular DCs for presentation to other B cells.
CD21 = CR2, expressed on B cells in noncovalent association with BCR. Can bind the C3b breakdown products; iC3b, C3d and C3dg. The close association allows the B cell to bind antigens via both BCR and CD21 simultaneously. This reduces the [Ag] necessary for B cell activatio.
CR3 and CR4 are important in the phagocytosis of complement-coated Ags. They both bind iC3b (= C3b broken down by complement factor I). CR3 also binds C3dg and factor H.
CRIg (macrophages resident in tissue) binds C3b. important in clearing of C3b opsonized Ag from the circulation (happens in liver).
C3aR, C5aR, C5L2 = GPCR. C3aR and C5aR mediate inflammatory funtions.
Complement enhances host defense
- formation of MAC
- opsonization
- inducing flammatory response that helps guide leukocytes to the site of infection.
MAC-induced cell death:
the large pore formation allows small molecules and ions to move through the membrane. the cell cannot retain osmotic integrety, and lyses.
If smaller pores are made, a type of apoptosis is induced after a calcium influx into the cytoplasm.
euk cells have many protective factors in their membranes to inactivate complement. However, in large enough numbers, MAC can overcome these.
Euk cells can recover from the MAC attack by shedding MAC-containing membrane vesicles out of the cell or by internalizing and degrading them.
Promotion of opsonization:
all the expected stuff, +
critical in viral infections, as the complement components can bind around the virus and prevent it from entering a cell.
Promotion of inflammation:
C3a and C5a are proinflammatory and chemoattractants for certain classes of leukocytes. bind to C3aR and C5aR on mast cells, macrophages, granulocytes, monocytes, endothelial cells, and some DCs. leads to the secretion of proinflammatory mediators like IL-6 and TNF-alpha (both facillitate leukocyte migration to said site).
