Microbiology
08-20-08
Complement System
Brian Bolerjack
51 minutos
1:
Complement (C) is part of the innate immune system that people love to hate. It has many
pathways and proteolytic cleavages.
2:
Complement is a group of sequentially reacting proteins which upon activation mediate a
number of biological reactions important to host defense. The Complement system is a cascade with
multiple activation events that lead to the generation of protein fragments which are important in the
host response. The complement system requires activation. That’s important because if it were
continually active it could cause autoimmune diseases. Normally, Complement is inactive (99.9% true).
The alternative pathway is always primed though.
3:
Nomenclature. The system was initially discovered 100 years ago, so it’s funky. Most proteins
start with the letter C. There are 11 different proteins, C1-C9, and then C1 is a complex of three proteins
and is broken into C1q, C1r, and C1s (each complex has one C1q and two C1r and C1s proteins). Most os
the “C” proteins, especially C1-C4, are part of the classical pathway of the complement system. C5-C9
form the terminal or lytic pathway of the complement system. Complement when fully activated has
the ability to lyse cells by punching holes in the cell membranes.
There are many proteins called factors. Factor B and D are critical components in the alternative
pathway. That’s probably the oldest of the three activation pathways. An overbar in older literature
indicates an active molecule (C1 with a bar over it).
Many proteins are cleaved. C3 is cleaved to C3b and C3a. Lowercase letters designate a
proteolytic fragment of a parent molecule. In almost all cases “a” means it is the smaller of the two
fragments. There is one exception, old literature uses the reverse for C2. C2a may be the larger
fragment, and C2b may be the smaller. The complement system uses receptors to mediate its biological
functions (except the terminal pathway). C3a binds to C3aR(eceptor). C5a binds to C5aR. C3a and C5a
are pro-inflammatory molecule and strong chemoattractants, so cells with the receptors for those
molecules can crawl along to sites of complement activation.
4:
Don’t memorize this slide. Out of the 40 proteins in the complement system, they can be
divided broadly into groups that make important biological points. Almost all of the proteins in the
activation of the complement system are serum soluble. The Mannose Binding Protein (MBP) or Ficolin
pathway has some homologues of the classical pathway, and they are involved in activation. There are a
lot of proteins involved in regulation. Some are serum soluble, and some are membrane bound. Many
are membrane bound and found on all cells in your body (with the exception of CR1). They prevent
complement mediated autologous(sp?) lysis, or complement damage of your own tissues, because
complement is nonspecific in its action (part of the innate immune system). That makes an important
biological point because half the proteins in the system are involved in regulation or preventing damage
to host tissues. Many receptors that mediate many functions of the system are also membrane bound.
5:
There are many family members quite related in the complement system. Many enzymes in the
activation pathways have a serine protease domain. C1s, C1r, and C2 in the classical pathway, and
Factors B and D in the alternative pathway, they are all very structurally similar molecules.
Collectins have a long collagen stalk and a globular head domain. C1q of the classical pathway,
and MBP and Ficolin of the additional activation pathway are all members. Proteins involved in the
membrane attack complex (MAC) have C9 or perforin domains. Perforin is the molecule that Cytotoxic T
cells and NK cells use to kill cells. Many regulatory molecules have the Short Consensus Repeat domain,
some of the membrane bound molecules like DAF and MCP are all composed of them, and can extend
Microbiology
Brian Bolerjack
08-20-08
51 minutos
Complement System
far from the cell surface through tandem repeats. “True” complement proteins C3, C4, and C5. They
are structurally related, C3 is the oldest. Most biological function mediated by complement are derived
from fragments when C3-5 are cleaved.
6:
Where is complement made? Main site is the hepatocytes of the liver, 90%. Remaining 10% is
made by many cell types. All cell types in the body can produce the regulatory molecules to protect
against complement damage, and also complement constitutively at low levels or when induced by
cytokines. Hematopoeitic cells, fibroblasts, endothelial cells, sperm and ova have receptors for
complement that may be involved in ligand receptor processes. Fat cells are the major producers of
Factor D, which is critical in the alternative pathway. Astrocytes and proteins can produce different
proteins and receptors. It’s clear that complement is made everywhere and is an important part of
innate immunity.
7:
These are the complement pathways. The three activation pathways, classical, Mannose
binding protein or ficolin pathway which shunts into the classical, and alternative. All the pathways
once activated function to generate enzymes that will cleave C3 and C5 into their main components C3a,
C3b, C5a and C5b, and C5b will self-associate with C6-9 which leads to the terminal or lytic pathway
which leads to the formation of the MAC.
8:
What do the molecules do and how are they generated? C3 when it’s cleaved, C3a is an
inflammatory mediator and chemoattractant. Cells with the C3aR like macrophages and neutrophils can
respond to the production of C3a. C3b is an important opsonin, it targets an invading pathogen for
phagocytosis and also neutralizes pathogens. C3b and its fragments are also important in B cell
activation, and is complement’s link to adaptive immune response, it helps to generate antibodies. C5a
is similar to C3a, but more potent, and is a chemoattractant and mediates inflammation. Many of its
functions mimic the acute phase response. C5b initiates formation of the MAC.
9:
What activates the complement system? Classical pathway is by Ag-Ab complexes, but not any
ol’ Abs. The best activator is IgM, and another good one is IgG. IgM is a pentamer in the blood. IgG is a
monomer. You need a minimum of two IgG molecules (at least two Fc regions) to activate the classical
pathway, and probably more than that. They need to be in close proximity on the surface of the Ag so
C1 can bridge the two. Mannose-Binding Protein pathway is activated by pathogens with Mannose of Nacetylglucosamine. Individuals with deficiencies in this pathway are very susceptible to these types of
pathogens. The alternative pathway is activated by LPS. (“95% sure this info will be on the boards”, so
might be one of his questions too, just my speculation).
10:
Classical pathway. C1 is part of the family of Collectins. The C1q portion has a collagen stalk and
6 globular heads. C1s and C1r kick off the classical pathway. Each C1 complex has two molecules each
of C1s and C1r.
11:
Video: Two IgG molecules bind epitopes on an Ag. C1 molecule bridges two close IgG molecules.
This causes a conformational change of C1q, which activates C1s and C1r. C1s cleaves C4 to C4a(no
function) and C4b, which can covalently attach to a pathogen through a thioester bond. C1s also cleaves
C2 to C2a and C2b. C4b associates with C2a and forms C4b2a, or the C3 convertase, the enzyme
complex that cleaves C3 in the classical pathway. C2a has the enzymatic activity and cleaves C3 to C3a
and C3b. Some of the C3b can associate with the C3 convertase, and it now becomes the C5 convertase
Microbiology
Brian Bolerjack
08-20-08
51 minutos
Complement System
(C4b2a3b), which wants to cleave C5. C2a of C5 convertase still has the enzymatic activity, and cleaves
C5 to C5a and C5b.
C5a and C3a are inflammatory mediators, so they activate many different cell types. They can
cause mast cells and basophils to degranulate, and chemoattract macrophages and neutrophils to the
site of complement activation to eat the bacteria. The C5b molecule starts formation of the MAC. It
binds to the membrane,C6,7,8,self associate- no more cleavages- and many C9 self associate with the
bound C5b678, and the C9 molecules form a pore in the membrane which lyses the cell. Complement is
everywhere throughout the body. Clay wanted me to include a pictogram to help you visual learners.
Classical pathway
Formation of the MAC
Microbiology
08-20-08
Complement System
Brian Bolerjack
51 minutos
12:
The alternative pathway is not as simple (his words). Through this process the complement
system is always a little bit activated. There is a process called C3 tickover. C3 in your blood is
continually being cleaved at a very low rate. If there is no pathogen present, the fragments that are
generated are shut down immediately by regulatory molecules of the complement system and nothing
happens. If there is a pathogen present, it recognizes it and the response is immediately started. It took
90 years to figure the pathway out somewhat.
Video: C3 also has a thioester bond, and when water interacts with it you get C3(H2O), and can
then bind Factor B. Factor D, a critical enzyme in the alternative pathway, cleaves Factor B to Ba and Bb.
Bb binds to C3H2O, and the initiation convertase of the alternative pathway is formed. C3(H2O)-Bb has
enzymatic activity in the Bb portion, and it cleaves C3 at a low rate to C3a and C3b. If there is a
pathogen present, C3b can covalently bind it like C4b could and mark it for elimination. C3b can also
bind Factor B, and Factor D then cleaves Factor B. C3b binds Bb and creates C3bBb, which is the actual
alternative pathway C3 convertase. The Bb portion of C3bBb can cleave lots of C3 to C3a and C3b,
which ramps up the process quickly. The new C3b molecules can go and bind to more pathogen, and
start the system over, or remain bound to the C3 convertase (now the alternative pathway C5
convertase). When C3b binds to C3bBb, its new affinity is for C5, and it can be cleaved to C5a and C5b
which then function in the same way as in the classical pathway. The alternative pathway is the more
ancient of the two pathways.
Alternative pathway
13:
Biological function and regulation. C3a and C5a are chemoattractants and anaphylatoxic
molecules. Because all cells have receptors for C3a and C5a, if there is a lot produced in your body you
can go into anaphylactic shock similar to that caused by allergies. They cause degranulation of mast
cells and basophils, which will release histamines, vasoactive amines and cytokines, which leads to an
increased vascular permeability and edema. That makes it easier for macrophages and neutrophils to
get into surrounding tissues to attack the pathogen. They can induce the production of cytokines like
TNF, IL-1, and IL-6. They can increase the expression of adhesion molecules and acute phase proteins.
They can activate neutrophils and macrophages to activate the respiratory burst, which is the
generation of free radicals to kill a phagocytosed pathogen.
Microbiology
08-20-08
Complement System
Brian Bolerjack
51 minutos
14:
C5b initiates the MAC. Some of the intermediates of the MAC might elicit signal transduction
for numerous cellular events. That may be important in protecting the host from self complement
damage.
15:
C3b is critical for the opsonization of Ag-Ab complexes for their clearance by macrophages and
neutrophils. It can bind covalently to the Ag or Ab. It is important for solublizing large immune
complexes which might otherwise collect together and precipitate out of solution and into tissues. C3b
helps break them up. This is what happens in diseases like serum sickness or Lupus, you get deposition
of complexes in tissues, activation of complement, and destruction of the tissue as a result. It also
neutralizes invading pathogens. It surrounds it and prevents it from attaching to any tissues.
16:
It has many potent effects and must be tightly regulated. It is regulated early on in activation of
the system and at the MAC complex. There are a few things that neutralize the effects of C5a and C3a
as well.
17:
Complement regulation is always in proportion to the amount of the activator present. One
bacteria won’t turn the whole system on, you will need 100,000 to do that. As the Ag is eliminated the
system won’t be continually activated. Another important regulation is limited half-life of the
convertases. Regulatory proteins will cleave the components of the convertases and shut them down
after a period of time. Naturally occurring inhibitory proteins control early in the pathway to keep it
from uncontrolled activation. There is a C1 inhibitor protein, a C4 binding protein. There are certain
carboxypeptidases that will cleave off the terminal arginine residue of C5a and C3a and that limits their
function. Some molecules regulate formation of the MAC.
18:
Decay Accelerating Factor (DAF) is an important membrane bound protein that will bind C3b
and prevent it from interacting with Factor B. Then you can’t make a C3 convertase. DAF can also push
Bb off the C3bBb complex to decrease its halflife. It can inhibit the formation or increase the decay of
the convertase.
19:
In the terminal pathway, there is a critical molecule called CD59, which binds to the forming C58 complex and prevent C9 from binding, thus the MAC cannot be fully formed. If C9 has bound
somewhat, it can prevent more C9 from binding and limit the MAC’s size.
20:
Deficiencies of the complement systems. If you have low activation component levels, you will
have recurrent bacterial infections. C1 (or C2) deficiency is associated with SLE and Loupus. They have
trouble handling immune complexes. If you are missing terminal components (fairly rare except with
C9), you will have recurrent bacterial infections. In the Japanese population, C9 deficiency is fairly
common, and they are most effected by nicerial infections. Missing regulatory components, like C1
inhibitor, you get HANE, Hereditary Angio Neurotic Edema. You get inflammation as the result of
trauma, but it is circumscribed and not in the normal manner. This can be treated by protein
replacement therapy. If you are missing DAF or CD59, those are linked to the membrane through GPI
anchors, you get Peroxismal Nocturnal Hemoglobinuria, where you get spontaneous lysis of RBCs
because they aren’t protected from complement mediated lysis. If you are missing receptors, like CR1,
that is associated with Lupus. A big problem is missing CR3 and CR4, you get big problems, and those
people usually die within the first few years of life.