Unit7: Complement System
Components and pathways of complement activation.
By-
Source: DEEPSHIKHA SAMDERSHI
Immunology DEPARTMENT OF ZOOLOGY
Kuby, 8th Edition MARWARI COLLEGE, RANCHI
• Complement was discovered many years ago (1890s; Jules Bordet) as a heat-labile (sensitive) component of blood
plasma that augments the opsonization of bacteria by antibodies and allows antibodies to kill some bacteria. This
activity was said to complement the antibacterial activity of antibody, hence the name.
• The complement system is made up of a large number of distinct plasma proteins that react with one another to
opsonize pathogens and induce a series of inflammatory responses that help to fight infection. These proteins are
present in form of inactive, precursor zymogens, widely distributed throughout body fluids and tissues without adverse
effect.
• The term complement refers to a set of 50-plus serum proteins that cooperates with both the innate and the adaptive
immune systems to eliminate pathogens, dying cells, and immune complexes from the body.
• Most complement components are synthesized in the liver by hepatocytes, although some are also produced by blood
monocytes, tissue macrophages, fibroblasts, and epithelial cells of the gastrointestinal and genitourinary tracts.
Complement components constitute approximately 15% of the globulin protein fraction in plasma.
Components of complement system:
(1) The complement pathways are initiated by proteins that
bind to pathogens, either directly or via an antibody or other
pathogen-specific protein. After a conformational change, (2)
enzymatic mediators activate other enzymes that generate the
central proteins of the complement cascade, the C3 and C5
convertases, which cleave C3 and C5, releasing active
components that mediate all functions of complement,
including (3) opsonization, (4) inflammation, and (5)
generation of the membrane attack complex (MAC). Effector
complement proteins can label an antibody-antigen complex for
phagocytosis (opsonins), initiate inflammation
(anaphylatoxins), or bind to a pathogen and nucleate the
formation of the MAC. Often, these effectors act through (6)
complement receptors on phagocytic cells, granulocytes, or
erythrocytes. (7) Regulatory proteins limit the
effects of complement by promoting their degradation or
preventing their binding to host cells.
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 sequence. Proteins that are inactive until cleaved by proteases are called zymogens.
Some complement proteases become active by binding to other macromolecules and undergoing a conformational
change; others are zymogens themselves, inactive until cleaved by another “upstream” protease. The two enzyme
complexes that cleave the complement components C3 and C5 are called the C3 and C5 convertases, respectively, and
occupy places of central importance in complement biology. The sequence of proteins in a complement pathway from the
initiator protein to the biological effector is referred to as a “complement cascade.”
3. Phagocytosis-enhancing components, or opsonins. On activation of the complement cascade, several complement
proteins are cleaved into two fragments, each of which then takes on a particular role. For C3 and C4, the larger
fragments, C3b and C4b, serve as opsonins, binding covalently to microbial cells and serving as ligands for phagocytic
cells with receptors for C3b or C4b.
4. Inflammatory mediators. Some small complement fragments act as inflammatory mediators. These fragments bind 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. Since these effects can be
harmful (even lethal) in excess, these fragments are called anaphylatoxins, derived from the Greek phrase meaning
“against protection.” C3a and C5a are examples of anaphylatoxins.
5. Membrane attack proteins. Proteins of the membrane attack complex (MAC) insert into the cell membranes of
invading microorganisms and punch holes that result in lysis of the pathogen. The complex itself forms a ring-shaped
multimer of complement proteins with a central hole through which cytoplasmic contents can escape. 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.
6. Complement receptor proteins. Receptor molecules on cell surfaces bind complement proteins and signal specific cell
functions. For example, some complement receptors such as CR1 bind to complement components such as C3b that have
opsonized pathogens, triggering phagocytosis of the C3b-bound pathogen. Binding of the anaphylatoxin complement
component C5a to C5a receptors (C5aRs) on neutrophils stimulates neutrophil degranulation and inflammation.
7. Regulatory complement components. Host cells are protected from unintended complement mediated
damage by the presence of regulatory proteins. These regulatory proteins include factor I, which degrades
C3b, and CD59 (protectin), which inhibits the formation of the MAC on host cells.
Pathways of Complement Activation:
There are three major pathways by which
the complement cascade can be initiated:
the classical pathway, the lectin
pathway, and the alternative pathway.
Although the initiating event of each of
the three pathways of complement
activation is different, they all converge in
the generation of an enzyme complex that
cleaves the C3 molecule. Enzymes that
cleave C3 into two fragments, C3a and
C3b, are referred to as C3 convertases.
The Classical Pathway Is Initiated by Antibody Binding to Antigens
The classical pathway of complement activation is considered part of the adaptive immune response since it begins with
the formation of antigen-antibody complexes. These complexes may be soluble, or they may be formed when an antibody
binds to antigenic determinants, or epitopes, situated on viral, fungal, parasitic, or bacterial cell membranes. Soluble
antibody antigen complexes are often referred to as immune complexes. Only complexes formed by antigens with
antibodies of the IgM class or certain subclasses of IgG antibodies are capable of activating the classical complement
pathway. The initial activation involves interaction of these antibody-antigen complexes with the complement
components C1, C2, and C4, normally found in the plasma as inactive precursors or zymogens.
The formation of an antigen-antibody complex induces conformational changes in the nonantigen-binding
(Fc) portion of the antibody molecule. This conformational change exposes a binding site on the antibody for the C1
component of complement. In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q and two
molecules each of the serine proteases C1r and C1s, held together in a Ca2+ -stabilized complex (C1qr2 s2 ).
• Each C1 macromolecular complex must bind to at least two antibody constant
regions for a stable C1q-antibody interaction to occur.
• In the serum, IgM exists as a pentamer of the basic four chain immunoglobulin
structure. In normal circulating, non-antigen bound condition, binding site for C1q
is not exposed. However, when pentameric IgM is bound to a multivalent antigen, it
undergoes a substantial conformational change that reveals at least three binding
sites for C1q. Thus, an IgM molecule engaged in an antibody-antigen complex can
bind C1q, whereas circulating, non-antigen bound IgM cannot.
• Monomeric IgG contains only one C1q-binding site per molecule. Although this C1q-binding site is exposed, the affinity of this
exposed binding site is too low to allow complement activation in the absence of antibody polymerization. When IgG antibodies bind
to their antigen, residues in the Fc portion of the antigen-complexed antibody participate in Fc-Fc binding to adjacent IgG molecules,
leading to the formation of IgG hexamers that bind C1q with high affinity and activate complement. Although smaller IgG complexes
are capable of some degree of complement activation, their lower affinity for C1q will correspondingly reduce the extent of
complement activity
• Apart from antigen-antibody complex,
C1q can bind to C-reactive protein
complexed with exposed
phosphocholine residues on bacteria.
It can also bind tissue damage
elements such as DNA, annexins A2
and A5, and histones on the surface of
apoptotic cells, resulting in
opsonization and engulfment. It also
appears to bind to as yet undefined
molecules on incomplete synapses,
facilitating their destruction.
• Binding of C1q to the Fc regions of the antigen-complexed antibody molecule induces a conformational change in one of
the C1r molecules. This conformational change in the C1r molecule converts it to an active serine protease enzyme that
then cleaves and activates its partner C1r molecule. The two C1r proteases then cleave and activate the two C1s
molecules.
• Activated C1s has two substrates, C4 and C2. The C4b fragment attaches covalently to the target membrane surface in
the vicinity of C1, and then binds C2.
• On binding C4b at the membrane surface or on an immune complex, C2 becomes susceptible to cleavage by the
neighboring C1s enzyme. A smaller C2b fragment diffuses away, leaving behind an enzymatically active C4b2a complex
(C3 convertase enzyme).
• The membrane-bound or immune complex–bound C3 convertase enzyme, C4b2a, now hydrolyzes C3, generating two
unequal fragments: the small anaphylatoxin C3a, and the pivotally important fragment C3b. A single C3 convertase
molecule can generate over 200 molecules of C3b, resulting in tremendous amplification at this step of the classical
pathway.
The generation of C3b is an essential precursor to many of the subsequent reactions of the complement system. Deficiencies of
complement components that act prior to C3 cleavage leave the host extremely vulnerable to both infectious and autoimmune diseases,
whereas deficiencies of components later in the pathway are generally of lesser consequence. In particular, patients with deficiencies in C3
itself are unusually susceptible to infections with both gram-positive and gram-negative bacteria.
C3b acts in three important and different ways to protect the host:
1. In a manner very similar to that of C4b, C3b binds covalently to microbial surfaces, providing a molecular “tag” that
allows phagocytic cells with C3b receptors to engulf the tagged microbes. This process is called opsonization.
2. C3b, like C4b, can attach to the Fc portions of antibodies participating in soluble antigen antibody complexes. These
C3b-tagged immune complexes are bound by C3b receptors on phagocytes or red blood cells, and are either
phagocytosed, or conveyed to the liver where they are destroyed.
3. Some molecules of C3b bind the membrane-localized C4b2a enzyme to form the trimolecular, membrane-bound, C5
convertase complex C4b2a3b. The C3b component of this complex binds C5, and the complex then cleaves C5 into the
two fragments: C5b and C5a. C4b2a3b is therefore the C5 convertase of the classical pathway.
The trio of tasks accomplished by the C3b molecule places it right at the center of complement attack pathways. C3b is
thus a central component in all three complement activation pathways.
The Lectin Pathway Is Initiated When Soluble Proteins Recognize Microbial Antigens
The lectin pathway of complement activation, like the classical pathway,
proceeds through the activation of a C3 convertase composed of C4b and
C2a. However, instead of relying on antibodies to recognize the microbial
threat and to initiate the complement activation process, this pathway uses
lectins—proteins that recognize particular carbohydrate components—as its
specific receptor molecules. Because it does not rely on antibodies from the
adaptive immune system, the lectin pathway is considered to be an arm of
innate, rather than adaptive, immunity.
• Receptor molecules: MBL (Mannose-binding Lectin), collectin-10 and
collectin-11 as well as several members of the ficolin family: ficolin-1,
ficolin-2, and ficolin-3.
Mannose-binding lectin (MBL), the first lectin demonstrated to be capable of
initiating complement activation, binds close-knit arrays of mannose (sugar)
residues that are found 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). It also recognizes N-acetylglucosamine, Dglucose, and L-
fucose polymers on microbial surfaces.
• In the blood, MBL is associated with MBL-associated serine proteases, or MASP proteins. Three MASP proteins—
MASP-1, MASP-2, and MASP-3—have been identified, but most studies of MASP function point to the MASP-2
protein as being the most important player in the next step of the MBL pathway.
• MASP-2 is structurally related to the serine protease C1s, and when MBL binds to a microbial surface, associated
MASP-2 molecules cleave both C2 and C4, giving rise to the C4b2a C3 convertase.
• The lectin pathway uses all the same downstream components as the classical pathway.
• In comparing the lectin and the classical pathways, we note that the soluble lectin receptor replaces the antibody as the
antigen-recognizing component, and MASP proteins take the place of C1r and C1s in cleaving C2 and C4, activating the
C3 convertase. Once the C3 convertase is formed, the reactions of the lectin pathway are the same as for the classical
pathway. The C5 convertase of the lectin pathway, like that of the classical pathway, is also C4b2a3b.
The Alternative Pathway Is Initiated in Three Distinct Ways
Initiation of the alternative pathway of complement activation, like the lectin pathway, is independent of antibody-
antigen interactions. Therefore, this pathway is also considered to be part of the innate immune system. However, unlike
the lectin pathway, the alternative pathway uses a different set of C3 and C5 convertases. The alternative pathway C3
convertase, C3bBb, is made up of one molecule of the C3b protein fragment and one molecular fragment unique to the
alternative pathway, Bb. A second C3b is then added to make the alternative pathway C5 convertase, C3bBbC3b.
Initiation of the alternative tickover pathway of
complement.
(a) Spontaneous hydrolysis of soluble C3 to C3(H2O)
allows the altered conformation of C3(H2O) to bind
factor B, rendering it susceptible to cleavage by factor
D. The resulting complex C3(H2O) Bb forms a fluid-
phase convertase capable of cleaving C3 to C3a and
C3b.
(b) Some of the C3b molecules formed by the fluid-phase
convertase bind to cell membranes. C3b, like C3(H2O),
binds factor B in such a way as to make B susceptible to
factor D–mediated cleavage.
(c) The membrane-bound C3bBb is stabilized by properdin
(factor P), which binds the C3bBb complex on the
membrane. Addition of a second C3b molecule to the
C3bBb complex forms the C5 convertase, which is also
stabilized by properdin.
The term tickover refers to the fact that C3 is constantly being made and spontaneously inactivated and is thus considered to
be “ticking over.” The alternative tickover pathway is initiated when C3, which is at high concentrations in serum,
undergoes spontaneous hydrolysis to yield the molecule C3(H2O). The conformation of C3(H2O) is different from that of
the C3 parent protein. C3(H2O) accounts for approximately 0.5% of plasma C3. In the presence of serum Mg2+, C3(H2O)
binds another serum protein, factor B. When bound to C3(H2O), factor B becomes susceptible to cleavage by a serum
protease, factor D. Factor D cleaves factor B, releasing a smaller Ba subunit that diffuses away, leaving a catalytically
active Bb subunit bound to C3(H2O). The C3(H2O) Bb complex is referred to as the “fluid-phase C3 convertase” because it
remains in the blood plasma and is not bound to any cells.
In the plasma, the fluid-phase convertase cleaves many molecules of C3 into C3a and C3b. The C3(H2O)Bb complex is not
very stable in a healthy host and it is rapidly degraded, hence the term “tickover” for this pathway.
There are two C3 convertases in the alternative tickover pathway: a fluid-phase C3(H2O)Bb, which initiates the pathway,
and a membrane-bound C3bBb C3 convertase that amplifies it and results in microbial destruction.
The cell-bound alternative pathway C3 convertase is unstable until it is bound by properdin (otherwise known as factor
P), a serum protein. Once stabilized by properdin, these cell-associated, C3bBb C3 convertase complexes rapidly
generate large numbers of C3b molecules on the microbial surface. Many of these then bind factor B, which is cleaved
in turn by factor D, thus facilitating the cleavage of yet more molecules of C3 and amplifying the rate of C3b
generation.
Just as the C5 convertase of the classical and lectin pathways was formed by the addition of C3b to the C4b2a C3
convertase complex, so the C5 convertase of the alternative pathway is formed by the addition of C3b to the alternative
pathway C3bBb C3 convertase complex. The alternative C5 convertase complex therefore has the composition
C3bBbC3b, and like the alternative C3 convertase, it is also stabilized by binding to properdin. Like the classical and
lectin pathway C5 convertase, C3bBbC3b cleaves C5, which goes on to form the MAC.
The Alternative Properdin-Activated Pathway
In the presence of Mg2+ , the immobilized properdin
bound C3b and factor B. This bound factor B proved to
be susceptible to cleavage by factor D, and the
resultant C3bPBb complex acted as an effective C3
convertase, leading to the amplification process.
Properdin can bind specifically to certain microbes, including Chlamydia pneumoniae, as well as to apoptotic and necrotic
cell surfaces. Once bound, properdin was indeed able to initiate the alternative pathway, as indicated above.
This pathway provides selective binding with the pathogens, rather than non-specific binding of C3b in tickover pathway.
This relies on pre-existence of low level of C3b.
The Alternative Protease-Activated Pathway
• The biochemical pathway that leads to complement activation is similar in concept to the blood coagulation pathway.
• Protein factors involved in blood clotting, such as thrombin, could cleave the complement components C3 and C5 ,
with the release of the active anaphylatoxins C3a and C5a.
• When blood platelets are activated during a clotting reaction, they release high concentrations of ATP and Ca2+ along
with serine/threonine kinases. These enzymes act to phosphorylate extracellular proteins, including C3b.
Phosphorylated C3b is less susceptible to proteolytic degradation than its unphosphorylated form, and thus, by this
route, activation of the clotting cascade enhances all of the complement pathways.
The Three Complement Pathways Converge at the Formation of C5 Convertase and
Generation of the MAC
All three initiation pathways culminate in the formation of C5
convertase. For the classical and lectin pathways, C5 convertase has
the composition C4b2a3b; for the alternative pathway, C5
convertase has the formulation C3bBbC3b. However, the end result of
all types of C5 convertase activity is the same: cleavage of the C5
molecule into two fragments, C5a and C5b.
The large C5b fragment is generated on the surface of the target cell or
immune complex and provides a binding site for the subsequent
components 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 C6.
Functions of Complement system:
• Lysis of bacterial and cell membranes
• Opsonization
• Induction of inflammation and chemotaxis by anaphylatoxins
• Augmentation of antibody responses
• Enhancement of immunologic memory
• Clearance of immune complexes from tissues
• Clearance of apoptotic cells
• Binding of complement components to antigen-presenting cells enhances their phagocytic ability and modulates
cytokine secretion.
• Complement components enhance the B cell–mediated immune response by increasing the avidity with which a B cell
binds to a complement-bound antigen.
• Immature T cells are protected from natural antibody and complement-mediated lysis by the provision of additional
sialic acid residues on their cell surface glycoproteins. Defective T cells do not have this protective layer, and so
complement participates in quality control mechanisms during T-cell development.
• Binding of C3a, C5a, and C3b to their respective receptors on mature T cells facilitates their growth, differentiation,
and survival.