2. • The term complement refers to a set of serum proteins that
cooperates with both the innate and the adaptive immune systems
to eliminate blood and tissue pathogens.
• Complement proteins interact with one another in catalytic cascades.
• Various complement components bind and opsonize bacteria,
rendering them susceptible to receptor-mediated phagocytosis by
macrophages, which express membrane receptors for complement
proteins.
• Other complement proteins elicit inflammatory responses, interface
with components of the adaptive immune system, clear immune
complexes from the serum, and/or eliminate apoptotic cells.
• Finally, a Membrane Attack Complex (MAC) assembled from
complement proteins directly kills some pathogens by creating pores
in microbial membranes.
4. • Research on complement began in the 1890s when Jules
Bordet at the Institut Pasteur in Paris showed that-
-sheep antiserum to the bacterium Vibrio cholerae
caused lysis (membrane destruction) of the bacteria, and
-heating the antiserum destroyed its bacteriolytic
activity.
-Surprisingly, the ability to lyse the bacteria was restored
to the heated serum by adding fresh serum that
contained no antibacterial antibodies.
• This finding led Bordet to reason that bacteriolysis
required two different substances:
– the heat-stable specific antibodies that bound to the
bacterial surface, and
– a second, heat-labile (sensitive) component responsible for
the lytic activity.
5. • In an effort to purify this second, nonspecific
component, Bordet developed antibodies specific
for red blood cells and
• used these antibodies, along with purified serum
fractions, to identify those fractions that
cooperated with the antibodies to induce lysis of
the red blood cells (hemolysis).
• The famous immunologist Paul Ehrlich, working
independently in Berlin, carried out similar
experiments and coined the term complement,
defining it as “the activity of blood serum that
completes the action of antibody.”
6. • The action of complement is the result of
interactions among a complex group of more
than 30 glycoproteins.
• Most complement components are
synthesized in the liver by hepatocytes,
although some are also produced by other cell
types, including blood monocytes, tissue
macrophages, fibroblasts, and epithelial cells
of the gastrointestinal and genitourinary
tracts.
7. • Complement components constitute
approximately 15% of the globulin protein
fraction in plasma, and their combined
concentration can be as high as 3 mg/ml.
• In addition, several of the regulatory components
of the system exist on cell membranes, so the
term complement therefore now embraces
glycoproteins distributed among the blood
plasma and cell membranes.
8.
9. Fig: Complement components can be classified into seven functional
categories:
• (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)
the 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.
10. 1. Initiator complement components.
• These proteins initiate their respective
complement cascades by binding to particular
soluble or membrane-bound molecules.
• Once bound to their activating ligand, they
undergo conformational alterations resulting in
changes in their biological activity.
• Examples of initiator complement components
are
– the C1q complex,
– Mannose Binding Lectin (MBL), and
– the ficolins
11. 2. Enzymatic mediators.
• Several complement components (e.g., C1r, C1s,
MASP2, and factor B) are proteolytic enzymes that
cleave and activate other members of the complement
cascade.
• Some of these proteases are activated by binding to
other macromolecules and undergoing a
conformational change.
• Others are inactive until cleaved by another protease
enzyme and are thus termed zymogens: proteins that
are activated by proteolytic cleavage.
• The two enzyme complexes that cleave complement
components C3 and C5, respectively, are called the C3
and C5 convertases and occupy places of central
importance in the complement cascades
12. 3. Membrane-binding components or opsonins.
• Upon activation of the complement cascade, several
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, enhancing phagocytosis by binding to
microbial cells and serving as binding tags for phagocytic
cells bearing receptors for C3b or C4b.
• As a general rule, the larger fragment of a cleaved
complement component is designated with the suffix
“b,” and the smaller with the suffix “a.”
• However, there is one exception to this rule: the larger,
enzymatically active form of the C2 component is named
C2a.
13. 4. Inflammatory mediators.
• Some small complement fragments act as
inflammatory mediators.
• These fragments enhance the blood supply to the area
in which they are released, by binding to receptors on
endothelial cells lining the small blood vessels and
inducing an increase in capillary diameter.
• They also attract other cells to the site of tissue
damage.
• Because such effects can be harmful in excess, these
fragments are called anaphylatoxins, meaning
substances that cause anaphylaxis (“against
protection”).
• Examples include C3a, C5a, and C4a.
14. 5. Membrane attack proteins.
• The 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 complement components of the MAC are
C5b, C6, C7, C8, and multiple copies of C9.
15. 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 on the
surface of pathogens, triggering phagocytosis of the
C3-bound pathogen.
• Binding of the complement component C5a to C5aR
receptors on neutrophils stimulates neutrophil
degranulation and inflammation.
• Complement receptors are named with “R,” such as
CR1, CR2, and C5aR.
16. 7. Regulatory complement components.
• Host cells are protected from unintended
complement-mediated lysis by the presence
of membrane-bound as well as soluble
regulatory proteins.
• These regulatory proteins include factor I,
which degrades C3b, and Protectin, which
inhibits the formation of the MAC on host
cells.
17. • There are three pathways of complement
activation
– The classical pathway
– The lectin pathway
– 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 capable of cleaving the C3
molecule into two fragments, C3a and C3b.
18. • The enzymes that accomplish this biochemical
transformation are referred to as C3 convertases.
• The classical and lectin pathways use the dimer
C4b2a for their C3 convertase activity;
• While the alternative pathway uses C3bBb to
achieve the same end
• However, the final result is the same: a dramatic
increase in the concentration of C3b, a centrally
located and multifunctional complement protein.
19. • The second set of convertase enzymes of the
cascade, C5 convertases, are formed by the
addition of a C3b component to the C3
convertases.
• C5 convertases cleave C5 into the
inflammatory mediator, C5a, and C5b, which is
the initiating factor of the MAC.
20.
21. FIGURE 6-2 The generation of C3 and C5 convertases by the three major pathways of
complement activation.
• The classical pathway is initiated when C1q binds to antigen-antibody complexes.
The antigen is shown here in dark red and the initiating antibody in green. The C1r
enzymatic component of C1 (shown in blue) is then activated and cleaves C1s,
which in turn cleaves C4 to C4a (an anaphylatoxin, bright red) and C4b. C4b
attaches to the membrane, and binds C2, which is then cleaved by C1s to form C2a
and C2b. (C2b is then acted upon further to become an inflammatory mediator.)
C2a remains attached to C4b, forming the classical pathway C3 convertase (C4b2a).
• In the lectin pathway, mannose binding lectin (MBL, green) binds specifically to
conserved carbohydrate arrays on pathogens, activating the MBL-associated serine
proteases (MASPs, blue). The MASPs cleave C2 and C4 generating the C3
convertase as in the classical pathway.
• In the alternative pathway, C3 undergoes spontaneous hydrolysis to C3(H2O), which
binds serum factor B. On binding to C3(H2O), B is cleaved by serum factor D, and
the resultant C3(H2O)Bb complex forms a fluid phase C3 convertase. Some C3b,
released after C3 cleavage by this complex, binds to microbial surfaces. There, it
binds factor B, which is cleaved by factor D, forming the cell-bound alternative
pathway C3 convertase, C3bBb. This complex is stabilized by properdin. The C5
convertases are formed by the addition of a C3b fragment to each of the C3
convertases.
22. The Classical Pathway Is Initiated by Antibody
Binding
• Considered part of the adaptive immune
response since it begins with the formation of
antigen-antibody complexes.
• These complexes may be soluble, that are often
referred to as immune complexes or
• These complexes may be formed when an
antibody binds to antigenic determinants, or
epitopes, situated on viral, fungal, parasitic, or
bacterial cell membranes.
• Only complexes formed by IgM or certain
subclasses of IgG antibodies are capable of
activating the classical complement pathway.
23. • The initial stage of activation involves the
complement components C1, C2, C3, and C4,
which are present in plasma as zymogens.
• The formation of an antigen-antibody complex
induces conformational changes in the non
antigen-binding (Fc) portion of the antibody
molecule.
• This conformational change exposes a binding
site for the C1 component of complement.
24. • 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
(C1qr2s2) .
• The C1q molecule itself is composed of 18
polypeptide chains that associate to form six
collagen-like triple helical arms, the tips of
which bind the CH2 domain of the antigen-bound
antibody molecule.
• Each C1 macromolecular complex must bind by
its C1q globular heads to at least two Fc sites for
a stable C1-antibody interaction to occur.
25.
26.
27. • The intermediates in the classical activation
pathway are depicted schematically in Figure
6-5.
• Note that binding of one component to the
next always induces either a conformational
change or an enzymatic cleavage, which
allows for the next reaction in the sequence to
occur.
28.
29. • The generation of C3b is centrally important to
many of the actions of complement.
• 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.
• This is because C3b acts in three important
and different ways to protect the host.
30. • First, C3b binds covalently to microbial surfaces,
providing a molecular “tag” and thereby allowing
phagocytic cells with C3b receptors to engulf the
tagged microbes. This process is called
opsonization.
• Second, C3b molecules 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.
31. • Finally, 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.
• This trio of tasks accomplished by the C3b molecule
places it right at the center of complement attack
pathways.
• C5b goes on to form the MAC with C6, C7, C8, and C9.
33. The Lectin Pathway Is Initiated When Soluble
Proteins Recognize Microbial Antigens
• The lectin pathway, like the classical pathway,
proceeds through the activation of a C3
convertase composed of C4b and C2a.
• This pathway uses lectins (proteins that
recognize specific carbohydrate components
primarily found on microbial surfaces) as its
specific receptor molecules
• Mannose-binding lectin (MBL)is the first lectin
demonstrated to be capable of initiating
complement activation,
34. • MBL binds close-knit arrays of mannose
residues that are found on microbial surfaces
such as those of Salmonella, Listeria, and
Neisseria strains of bacteria; Cryptococcus
neoformans and Candida albicans strains of
fungi; and even the membranes of some
viruses such as HIV-1 and respiratory syncytial
virus.
• The complement pathway that it initiates is
referred to as the lectin pathway of
complement activation.
35.
36. • MBL also recognizes structures in addition to
mannose, including N-acetyl glucosamine, D-glucose,
and L-fucose.
• Thus, MBL acts as a classic pattern recognition
receptor
• Many individuals with low levels of MBL suffer from
repeated, serious bacterial infections.
• MBL is constitutively expressed by the liver and, like
C1q, MBL belongs to the subclass of lectins known as
collectins (MBL structurally resembles C1q )
• More recently, the ficolins, another family of proteins
structurally related to the collectins, have been
recognized as additional initiators of the lectin
pathway of complement activation.
• L-ficolin, H-ficolin, and M-ficolin each bind specific
types of carbohydrates on microbial surfaces.
37. • MBL is associated in the serum with MBL-
Associated Serine Proteases, or MASP proteins.
• Three MASP proteins—MASP1, MASP2, and
MASP3—have been identified, MASP2 protein
being most important in the next step of the
MBL pathway.
• MASP-2 is structurally related to the serine
protease C1s, and can cleave both C2 and C4,
giving rise to the C3 convertase, C4b2a
• Thus, the lectin pathway utilizes all the same
components as the classical pathway with the
single exception of the C1 complex.
38. • The soluble lectin receptor replaces the
antibody as the antigen-recognizing
component, and
• MASP proteins take the place of C1r and C1s
in cleaving and 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.
39.
40. The Alternative Pathway Is Initiated in Three Distinct
Ways
• Initiation of the alternative pathway of complement
activation is independent of antibody-antigen
interactions, and
• So this pathway, like the lectin pathway, is also
considered to be part of the innate immune system.
• However, unlike the lectin pathway, the alternative
pathway uses its own set of C3 and C5 convertases.
• The alternative pathway C3 convertase is made up of
one molecule of C3b and one molecule unique to the
alternative pathway, Bb.
• A second C3b is then added to make the alternative
pathway C5 convertase.
41. • The alternative pathway can itself be initiated in
three distinct ways.
• The first mode of initiation to be discovered, the
“tickover” pathway, utilizes the four serum
components C3, factor B, factor D, and properdin
– The term tickover refers to the fact that C3 is
constantly being made and spontaneously inactivated
and is thus undergoing “tickover.”
• Two additional modes of activation for the
alternative pathway have also been identified:
– one is initiated by the protein properdin, and
– the other by proteases such as thrombin and
kallikrein.
42. The Alternative Tickover Pathway
• The alternative tickover pathway is initiated when
C3, which is at high concentrations in serum,
undergoes spontaneous hydrolysis at its internal
thioester bond, yielding the molecule C3(H2O).
• The conformation of C3(H2O) has been
demonstrated to be different from that of the
parent protein, C3.
• C3(H2O) accounts for approximately 0.5% of
plasma C3 and, in the presence of serum Mg2+
binds another serum protein, factor B (Figure 6-
8a).
• When bound to C3(H2O), factor B becomes
susceptible to cleavage by a serum protease,
factor D.
43. • Factor D cleaves B, releasing a smaller Ba subunit,
which diffuses away and leaves a catalytically
active Bb subunit that remains bound to C3(H2O).
• This C3(H2O)Bb complex, which at this point is
still in the fluid phase (i.e., in the plasma, not
bound to any cells), has C3 convertase activity. It
rapidly cleaves many molecules of C3 into C3a
and C3b (Figure 6-8b).
• This initiating C3 convertase is constantly being
formed in plasma, and breaking down a few C3
molecules, but it is then just as rapidly degraded.
44.
45. • However, if there is an infection present, some
of the newly formed C3b molecules bind nearby
microbial surfaces via their thioester linkages.
• Since factor B is capable of binding to C3b as
well as to C3(H2O),
– factor B now binds the newly attached C3b
molecules on the microbial cell surface (see Figure
6-8b), and
– again becomes susceptible to cleavage by factor D,
with the generation of C3bBb complexes.
• These C3bBb complexes are now located on the
microbial membrane surface.
46. • Like the C4b2a complexes of the classical
pathway, the cell-bound C3bBb complexes have
C3 convertase activity, and
• This cell-bound C3bBb complex now takes over
from the fluid phase C3(H2O)Bb as the
predominant C3 convertase.
• To be clear, there are two C3 convertases in the
alternative tickover pathway:
– a fluid phase C3(H2O)Bb, which kicks off the pathway,
and
– a membrane-bound C3bBb C3 convertase.
47.
48. • The cell-bound C3bBb C3 convertase is unstable
until it is bound by properdin, a protein from the
serum (Figure 6-8c).
• Once stabilized by properdin, these rapidly
generate large amounts of C3b at the microbial
surface;
• These C3b, in turn, bind more factor B, facilitating
its cleavage and activation and resulting in a
dramatic amplification of the rate of C3b
generation.
• This amplification pathway is rapid and, once the
alternative pathway has been initiated, more
than 2 x 106 molecules of C3b can be deposited
on a microbial surface in less than 5 minutes.
49. • The C5 convertase of the alternative pathway
is formed by the addition of C3b to the
alternative pathway C3 convertase complex.
• The C5 convertase complex therefore has the
composition C3bBbC3b, and, like the C3
convertase, is also stabilized by binding to
factor P.
• C5 convertase, C3bBbC3b cleaves C5, which
goes on to form the MAC
50. • The end result of all types of C5 convertase activity
is the same: the 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 MAC.
• However, the C5b component
– Is extremely labile as its not covalently bound to the
membrane like C3b and C4b, and
– Is rapidly inactivated unless it is stabilized by the
binding of C6.
51. • Up to this point in the complement cascades,
all of the complement reactions take place on
the hydrophilic surfaces of microbes or on
immune complexes in the fluid phase of blood,
lymph, or tissues.
• In contrast, when C5b binds C6 and C7, the
resulting complex undergoes a conformational
change that exposes hydrophobic regions on
the surface of the C7 component capable of
inserting into the interior of the microbial
membrane.
52. • C8 is made up of two peptide chains: C8β and
C8αϒ.
• Binding of C8β to the C5b67 complex induces a
conformational change in the C8 dimer such that
the hydrophobic domain of C8αϒ can insert into
the interior of the phospholipid membrane.
• The C5b678 complex can create a small pore, 10
Å in diameter, and formation of this pore can lead
to lysis of red blood cells, but not of nucleated
cells.
• The final step in the formation of the MAC is the
binding and polymerization of C9 to the C5b678
complex.
53. • As many as 10 to 19 molecules of C9 can be
bound and polymerized by a single C5b678
complex.
• During polymerization, the C9 molecules undergo
a transition, so that they, too, can insert into the
membrane.
• The completed MAC, which has a tubular form
and functional pore diameter of 70 Å to 100 Å,
consists of a C5b678 complex surrounded by a
poly-C9 complex.
• Loss of plasma membrane integrity leads
irreversibly to cell death.
