THE

COMPLEMENT SYSTEM
MLC 402 – IMMUNOLOGY

DR. K. S. AKIINWANDE
THE COMPLEMENT SYSTEM
◦ Is the major effector of the humoral branch of the immune system
◦ History - Jules Bordet at the Pasteur Institute in the 1890s demonstrated that sheep
antiserum to the bacterium Vibrio cholerae caused lysis of the bacteria and that heating the
antiserum destroyed its bacteriolytic activity.
◦ Upon adding antibody - free fresh serum, ability to lyse the bacteria was restored to the
heated serum. Bordet correctly reasoned that the heat-sensitive component was responsible
for the lytic activity.
◦ Paul Ehrlich in Berlin independently carried out similar experiments and coined the term
complement, defining it as “the activity of blood serum that completes the action of antibody.”
◦ Researchers later discovered that the action of complement was the result of interactions of a
large and complex group of protein
The Functions of Complement
◦ Lysis of cells, bacteria, and viruses
◦ Opsonization, which promotes phagocytosis of particulate antigens
◦ Binding to specific complement receptors on cells of the immune system,
triggering specific cell functions, inflammation, and secretion of
immunoregulatory molecules
◦ Immune clearance, which removes immune complexes from the circulation
and deposits them in the spleen and live
Components of the Complement System
◦ The proteins and glycoproteins that compose the complement system are
synthesized mainly by liver hepatocytes.
◦ Significant amounts are also produced by blood monocytes, tissue macrophages,
and epithelial cells of the gastrointestinal and genitourinary tracts.
◦ Most circulate in the serum in functionally inactive forms as proenzymes, or
zymogens, which are inactive until proteolytic cleavage, which removes an inhibitory
fragment and exposes the active site.
◦ The complement-reaction sequence starts with an enzyme cascade
Components of the Complement System
◦ Complement components are designated by numerals (C1–C9), by letter symbols (e.g., factor
D), or by trivial names (e.g., homologous restriction factor).
◦ Peptide fragments formed by activation of a component are denoted by small letters.
◦ In most cases, the smaller fragment resulting from cleavage of a component is designated “a”
and the larger fragment designated “b” (e.g., C3a, C3b; note that C2 is an exception: C2a is the
larger cleavage fragment).
◦ The larger fragments bind to the target near the site of activation, and the smaller fragments
diffuse from the site and can initiate localized inflammatory responses by binding to specific
receptors.
◦ The complement fragments interact with one another to form functional complexes. Those
complexes that have enzymatic activity are designated by a bar over the number or symbol
(e.g., C4b2a, C3bBb)
Complement Activation
◦ Activation of complement can occur through 3 pathways:
◦ the classical pathway,
◦ the alternative pathway, or
◦ the lectin pathway.
◦ The early steps, through which any of the 3 pathways can proceed, all
culminates in formation of C5b,
◦ The final steps from C5b, leading to formation of a membrane attack are all
the same pathways
The Classical Pathway
◦ Complement activation by the classical pathway commonly begins with the formation of soluble .
antigen-antibody complexes (immune complexes) or with the binding of antibody to antigen on a
suitable target, such as a bacterial cell.
◦ The initial stage of activation involves C1, C2, C3, and C4, which are present in plasma in functionally
inactive forms.
◦ C1 in serum is a macromolecular complex consisting of C1q and two molecules each of C1r and C1s, held
together in a complex (C1qr2s2) stabilized by Ca2+ ions
◦ IgM and certain subclasses of IgG (human IgG1, IgG2, and IgG3) can activate the classical complement
pathway.
◦ The formation of an antigen-antibody complex induces conformational changes in the Fc portion of the
IgM molecule that expose a binding site for the C1 component of the complement system.
◦ Each C1 molecule must bind by its C1q globular heads to at least two Fc sites for a stable C1-antibody
interaction to occur. When pentameric IgM is bound to antigen on a target surface it assumes the so-
called “staple” configuration, in which at least three binding sites for C1q are exposed.
The Classical Pathway
◦ An IgG molecule, on the other hand, contains only a single C1q-binding site in the CH2 domain of the Fc, so
that firm C1q binding is achieved only when two IgG molecules are within 30–40 nm of each other on a target
surface or in a complex, providing two attachment sites for C1q.
◦ Binding of C1q to Fc binding sites induces a conformational change in C1r activates C1r to an active serine
protease enzyme, C1r, which in turn activates C1s to a similar active enzyme, C1s.
◦ C1s has two substrates, C4 and C2.
◦ C1s hydrolyzes the polypeptide chains and eventual fragmentation of C4, leading to small fragment -C4a and
the larger fragment - C4b.
◦ The smaller fragment from C4 cleavage, C4a, together with other smaller fragments of C4, C3, and C5 do not
participate directly in the complement cascade, rather, they are anaphylatoxins - mediator of inflammation.
◦ The C4b fragment attaches to the target surface in the vicinity of C1, and conversely binds the C2. C2 is then
cleaved by the neighboring C1s to C2a and C2b. The larger fragment binds to C4b while the smaller fragment
(C2b) diffuses away.
◦ The resulting C4b2a complex is called C3 convertase, referring to its role in converting the C3 into an active
form.
The Classical Pathway
The Classical Pathway
◦ The C3 convertase hydrolyses the native C3 component to generate the short fragment - C3a
– and the larger fragment - C3b.
◦ A single C3 convertase molecule can generate over 200 molecules of C3b, resulting in
tremendous amplification at this step of the sequence.
◦ Some of the C3b binds to C4b2a to form a trimolecular complex C4b2a3b, called C5
convertase.
◦ The C3b component of this complex binds C5 and the C4b2a component can cleave C5 into
C5a, which diffuses away, and C5b, which attaches to C6 and initiates formation of the
membrane attack complex (MAC)
◦ Some of the C3b generated by C3 convertase activity does not associate with C4b2a instead
it diffuses away and then coats immune complexes and particulate antigens, functioning as
an opsonin, or can remain bound to cell membranes.
The Classical Pathway
The Alternative Pathway
– Ab independent
◦ The alternative pathway generates bound C5b, the same product that the classical pathway
generates, but it does so without the need for antigen-antibody complexes for initiation.
◦ Because no antibody is required, the alternative pathway is a component of the innate
immune system.
◦ This major pathway of complement activation involves four serum proteins: C3, factor B,
factor D, and properdin.
◦ The alternative pathway is initiated in most cases by cell-surface constituents that are
foreign to the host. For example, both gram-negative and gram-positive bacteria have cell-
wall constituents that can activate the alternative pathway.
◦ In the classical pathway, C3 is rapidly cleaved to C3a and C3b by the enzymatic activity of the
C3 convertase. In the alternative pathway, serum C3, which contains an unstable thioester
bond, is subject to slow spontaneous hydrolysis to yield C3a and C3b.
 The Alternative Pathway
– Ab independent
◦ The C3b present on the surface of the foreign cells can bind another serum protein called factor
B to form a complex stabilized by Mg2+.
◦ Binding of C3b to factor B allows the enzymatically active serum protein called factor D to cleave
the C3b-bound factor B, releasing a small fragment - Ba that diffuses away and generating
C3bBb.
◦ The C3bBb complex has C3 convertase activity and thus is analogous to the C4b2a complex in
the classical pathway.
Footnote:
The C3b component can bind to foreign surface antigens (such as those on bacterial cells or viral particles) or even
to the host’s own cells. The membranes of most mammalian cells have high levels of sialic acid, which contributes
to the rapid inactivation of bound C3b molecules on host cells.
But because many foreign antigenic surfaces (e.g., bacterial cell walls, yeast cell walls, and certain viral envelopes)
have only low levels of sialic acid, C3b bound to these surfaces remains active for a longer time.
The Alternative Pathway
– Ab independent
◦ The C3 convertase activity of C3bBb has a half-life of only 5 minutes unless
the serum protein properdin binds to it, stabilizing it and extending the half-
life of this convertase activity to 30 minutes.
◦ The C3bBb generated in the alternative pathway can activate unhydrolyzed
C3 to generate more C3b autocatalytically.
◦ The C3 convertase activity of C3bBb generates the C3bBb3b complex, which
exhibits C5 convertase activity, analogous to the C4b2a3b complex in the
classical pathway.
◦ The nonenzymatic C3b component binds C5, and the Bb component
subsequently hydrolyzes the bound C5 to generate C5a and C5b.
The Alternative Pathway
– Ab independent

C5
The Lectin Pathway
◦ The Lectin Pathway originates with host proteins binding microbial surfaces
◦ Lectins are proteins that recognize and bind to specific carbohydrate targets.
◦ The lectin pathway, like the alternative pathway, does not depend on antibody for its
activation. However, the mechanism is more like that of the classical pathway,
because after initiation, it proceeds, through the action of C4 and C2, to produce a
C5 convertase.
◦ The lectin pathway is activated by the binding of mannose-binding lectin (MBL) to
mannose residues on glycoproteins or carbohydrates on the surface of
microorganisms including certain Salmonella, Listeria, and Neisseria strains, as well
as Cryptococcus neoformans and Candida albicans.
◦ MBL is an acute phase protein produced in inflammatory responses. Its function in
the complement pathway is similar to that of C1q, which it resembles in structure.
The Lectin Pathway
◦ After MBL binds to the surface of a cell or pathogen, MBL-associated serine
proteases, MASP-1 and MASP-2, bind to MBL.
◦ The active complex formed by this association causes cleavage and activation
of C4 and C2. The MASP-1 and -2 proteins have structural similarity to C1r
and C1s and mimic their activities.
◦ This means of activating the C2–C4 components to form a C5 convertase
without need for specific antibody binding represents an important innate
defense mechanism comparable to the alternative pathway, but utilizing the
elements of the classical pathway except for the C1 proteins.
 The Membrane Attack Complex (MAC)
◦ The terminal sequence of complement activation involves C5b, C6, C7, C8, and C9, which interact sequentially to
form a macromolecular structure called the membrane-attack complex (MAC).
◦ This complex forms a large channel through the membrane of the target cell, enabling ions and small molecules to
diffuse freely across the membrane.
◦ The end result of activating the classical, alternative, or lectin pathways is production of an active C5 convertase.
◦ The C5b component is extremely labile and becomes inactive within 2 minutes unless C6 binds to it and stabilizes its
activity.
◦ As C5b6 binds to C7, the resulting complex undergoes a hydrophilic-amphiphilic structural transition that exposes
hydrophobic regions, which serve as binding sites for membrane phospholipids.
◦ If the reaction occurs on a target-cell membrane, the hydrophobic binding sites enable the C5b67. Released C5b67
complexes can insert into the membrane of nearby cells and mediate “innocent-bystander” lysis.
◦ Binding of C8 to membrane-bound C5b67 induces a conformational change in C8, so that it too undergoes a
hydrophilic-amphiphilic structural transition. The C5b678 complex creates a small pore, which can lead to lysis of red
blood cells but not of nucleated cells.
◦ The final step in formation of the MAC is the binding and polymerization of C9, a perforin-like molecule, to the
C5b678 complex. As many as 10–17 molecules of C9 can be bound and polymerized by a single C5b678 complex.
 The Membrane Attack Complex (MAC)
◦ The final step in formation of the MAC is the binding and polymerization of C9, a perforin-like molecule, to the
C5b678 complex. As many as 10–17 molecules of C9 can be bound and polymerized by a single C5b678
complex.
Regulation of the Complement
System
◦ Because many elements of the complement system are capable of attacking host cells as well as foreign cells
and microorganisms, elaborate regulatory mechanisms have evolved to restrict complement activity to
designated targets.
◦ A general mechanism of regulation in all complement pathways is the inclusion of highly labile components
that undergo spontaneous inactivation if they are not stabilized by reaction with other components.
◦ In addition, a series of regulatory proteins can inactivate various complement components. For example, the
glycoprotein C1 inhibitor (C1Inh) can form a complex with C1r2s2, causing it to dissociate from C1q and
preventing further activation of C4 or C2.
◦ The potential destruction of healthy host cells by C3b is further limited by a family of related proteins that
regulate C3 convertase activity in the classical and alternative pathways.
◦ These regulatory proteins all contain repeating amino acid sequences (or motifs) of about 60 residues,
termed short consensus repeats (SCRs). All these proteins are encoded at a single location on chromosome 1
in humans, known as the regulators of complement activation (RCA) gene cluster.