Complement systems:

Complement system and their biological function

(Classical and Alternative pathway)

Objectives:

After carefully listening to this lecture, you will be able to:

 Define complement system

 Know the different complement system
 Understand the component of the complement system
 Explain how different pathways are activated
 Know the difference in the pathways of the different complement system

Introduction:

The complement system is a part of the immune system that enhances

(complements) the ability of antibodies and phagocytic cells to clear microbes and
damaged cells from an organism, promotes inflammation, and attacks the
pathogen's plasma membrane. It is part of the innate immune system, which is not
adaptable and does not change over the course of an individual's lifetime. It can be
recruited and brought into action by the adaptive immune system. It consists of a
number of small proteins found in the blood, in general synthesized by the liver,
and normally circulating as inactive precursors (pro-proteins).It contributes to
inflammation by inducing local changes in blood flow and the influx of
inflammatory cells into the affected area. When stimulated by one of several
triggers, proteases in the system cleave specific proteins to release cytokines and
initiate an amplifying cascade of further cleavages. The pathology that
accompanies uncontrolled activation or incomplete performance of complement’s
function is often the result of a deficiency or impairment of one of the components.

Overview:
Complement system are mainly activated by three main pathway i.e. classical
complement pathway, alternative complement pathway and lectin pathway. Each
complement pathway has unique proteins for the initiating steps, but shared the
same or related proteins for the intermediate steps and uses the same components
in the last step, culminating in the same activities. Each initiating pathway is
triggered by a different type of activator, usually a cell, microbe, or molecular
aggregate that presents charge patterns that are “recognized” by component of the
individual initiating pathway, making complement one of the innate immune
system’s primary pattern-recognition mechanisms for detecting nonself. They are
triggered by 1) the binding of one of their components to the activator, 2) a cascade
of enzyme activation, and 3) generation of biological effects. There are physical
and chemical mechanisms as well as specific regulators that prevent uncontrolled
activation and damage to local cells and tissues. A network of fluid-phase and cell
associated regulatory proteins and specific receptors interact with the component
of complement and their split products, and are involved in controlling
complement activation at the cell surface, as well as a wide variety of cell-
signaling events.

Functions of complement:

After initial activation, the various complement components interact, in a highly

regulated cascade, to carry out a number of basic functions including:

 Lysis of cells, bactera, 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 liver

Complement components:

The components of complements (proteins and glycoproteins) are synthesized by

liver hepatocytes, blood monocytes, tissue macrophages, and epithelial cells of the
gastrointestinal and genitourinary tracts. They are circulated in the serum in the
form of proenzymes, or zymogens. The complement-reaction sequences starts with
an enzyme cascade.

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 fragments resulting from cleavage of a component is
designated “a” and the larger fragment designated “b” (e.g. C3a, C3b but C2 is an
exception: C2a is the larger fragment). 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)

Complement activation:

There are three pathways that can activate the complement system, and different
structural areas of the immunoglobulin molecules are involved in complement
fixation by two of them. Complement is the name given to a complex series of
some 20 proteins which, along with blood clotting, fibrinolysis and kinin formation
forms one of the triggered enzymes system found in plasma. The final step of the
three pathways is the formation of membrane attack complex (MAC) in all the
pathways.

The classical pathway of complement activation:

Complement activation by the classical pathway commonly begins with the
formation of soluble antigen-antibody complexes or with the binding of antibody
to antigen on a suitable target, such as bacterial cell. The initial stage of activation
involves C1, C2, C3 and C4 which are present in plasma in functionally inactive
forms.

The formation of an antigen-antibody complex induces conformational changes in

the Fc portion of the IgM molecules that expose a binding site for the C1
component of the complement system. C1 in serum is a macromolecular complex
consisting of C1q and the two molecules each of C1r and C1s, held together in a
complex(C1qr2s2) stabilized by Ca2+ ions. Each C1r and C1s monomer contains a
catalytic domain and an interaction domain, the latter facilitates interaction with
C1q or with each other.

When the 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. Binding of C1q to Fc binding sites induces a conformational change in
C1r that convert C1r to an active serine protease enzyme, C1r, which then cleaves
C1s to a similar active enzyme, C1s. C1s has two substrates C4 and C2. The C4
component is a glycoprotein containing three polypeptide chain α,β and γ. C4 is
activated when C1s hydrolyze a small fragment (C4a) from the amino terminus of
the α-chain exposing a binding site on the larger fragment (C4b). The C4b
fragment attaches to the target surface in the vicinity of C1 and the C2 proenzyme
then attaches to the exposed binding site on C4b. where the C2 is then cleaved by
the neighbouring C1s; 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 smaller fragment from C4 cleavage, C4a, is an
anaphylatoxin, or mediator of inflammation, which does not participate directly in
the complement cascade.

The native C3 component consists of the two polypeptide chains, α and β.

Hydrolysis of a short fragment (C3a) from the amino terminus of the α-chain by
the C3 convertase generates C3b. some of the C3b binds to C4b2a to form a
trimolecular complex C4b2a3b, called C5 convertage. The C3b component of this
complex bind C5 and alters its conformation, so that the C4b2a component can
cleave C5 into C5a, which diffuses away, and C5b, which attaches to C6 and
initiates formation of the menbrane –attack complex(MAC).

Alternative pathway:

The alternative 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. Here serumC3, which
contains an unstable thioester bond, is subject to slow spontaneous hydrolysis to
yield C3a and C3b. The C3b component can bind to foreign surface antigens or
even to the host’s own cells. But the C3b bound to the bacterial cell wall remains
active for a longer time. 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 exposes a site on factor B that serves as the substrate for an
enzymatically active serum protein called factor D. factor D cleaves 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. The C3 convertase activity of C3bBb
has a half-life of only 5minutes 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 actiavate unhydrolyzed C3 to

generate more C3b autocatalytically. As a result, the initial step are repeated and
amplified, so that more than 2 X 106 molecules of C3b can be deposited on an
antigenic surface in less than 5 minutes. The c3 convertase activity of C3bBb
generates the C3bBb3b complex, which exhibit 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 genetates C5a and C5b; the latter binds to the antigenic surfaces.

The lectin pathway:

Lectins are proteins that recognize and bind to specific carbohydrate targets. It
does not depend on antibody for its activation. However, the mechanism is more
like that of the classical pathway because after its 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. MBL is an acute phase protein produce in inflamatary responses.
Its function in the complement pathway is similar to that of C1q, which it
resembles in structure. After MBL binds to the surface of the 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 MASP-2 have structurally similar 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 defence mechanism.
Differences between the complement pathways:

The classical pathway is activated, for example, by Ag–Ab complexes that react
with activated C1q. The lectin pathway is initiated by either serum MBL or ficolins
that recognize certain oligosaccharide moieties on microbial surfaces. The
alternative pathway can be activated either by the presence of foreign surfaces such
as LPSs or through C3b generated by spontaneous hydrolyses, the so-called “tick-
over”. All three pathways merge at the level of C3, and activation of either
pathway ultimately results in generation of the potent proinflammatory
complement split products C3a and C5a as well as the terminal membrane attack
complex (MAC).

Biological functions of complement system:

Complement serves as an important mediator of the humoral response by

amplifying the response and converting it into an effective defense mechanism to
destroy invading microorganisms. The MAC mediates cell lysis, while other
complement components or split products participate in the inflammatory
response, opsonization of antigen, viral neutralization, and clearance of immune
complexes.

The membrane-attack complex formed by complement activation can lyse gram-

negative bacteria, parasites, viruses, erythrocytes, and nucleated cells. These
pathways serve as an important innate immune defense against infectious
microorganisms as the alternative and lectin pathways of activation generally occur
without an initial antigen-antibody interaction. The requirement for an initial
antigen-antibody reaction in the classical pathway supplements these nonspecific
innate defenses with a more specific defense mechanism. The complement system
is quite effective in lysing gram-negative bacteria but resistant in gram-positive
bacteria because the thick peptidoglycan layer in their cell wall prevents insertion
of the MAC into the inner membrane.

The cleavage products of complement components can mediate inflammation. The

smaller fragments resulting from complement cleavage, C3a, C4a and C5a, called
anaphylatoxins, bind to receptors on mast cells and blood basophils and induce
degranulation, with release of histamine and other pharmacologically active
mediators.

The complement system mediates viral neutralization by a number of mechanisms.

The binding of antibody and/ or complement to the surface of a viral particle
creates a thick protein coating which neutralizes viral infectivity by blocking
attachment to susceptible host cells. The deposits of antibody and complement on
viral particles also facilitate binding of viral particle to cells possessing Fc or type
1 complement receptor (CR1). Finally, complement is effective in lysing most,
enveloped viruses resulting in fragmentation of the envelope and disintegration of
the nucleocapsid.

Conclusion:

From the above study we learnt that complement system is an important immune
system, which help us to degrade the antigen and protect our body from the
harmful effects of antigens. They can eliminate foreign particles through various
ways as we studied earlier. Their active participation in immune system is very
important. Deficiency in any of the protein associated with the complement system
can lead to various diseases such as asthma , glomerulonephritis, various forms
of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel
disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic
syndrome and ischemia-reperfusion injuries, and rejection of transplanted organs.
The complement system is also becoming increasingly implicated in diseases of
the central nervous system such as Alzheimer's disease and other
neurodegenerative conditions such as spinal cord injuries.

Glossary:

1. alternative pathway (of complement activation): Activation pathway

involving complement components C3, Factor B, Factor D, and Properdin
which, in the presence of a stabilizing activator surface such as microbial
polysaccharide, generates the alternative pathway C3 convertase C3bBb.
2. anaphylatoxin: A substance (e.g. C3a, C4a or C5a) capable of directly
triggering mast cell degranulation.
3. Complement: A group of serum proteins, some of which act in an
enzymatic cascade, producing effector molecules involved in inflammation
(C3a, C5a), phagocytosis (C3b), and cell lysis (C5b-9).
4. Classical pathway (of complement activation): Activation pathway
involving complement components Cl, C2 and C4 which, following fixation
of Clq, e.g. by antigen-antibody complexes, produces the classical pathway
C3 convertase C4b2a.
5. Cytotoxic: Kills cells.
6. Innate immunity: Immunity which is not intrinsically affected by prior
contact with antigen, i.e. all aspects of immunity not directly mediated by
lymphocytes.
7. Lectins: A family of proteins, mostly of plant origin, which bind specific
sugars on glycoproteins and glycolipids. Some lectins are mitogenic (e.g.
PHA, ConA).
8. Macrophage: Large phagocytic cell, derived from the blood monocyte,
which also functions as an antigenpresenting cell and can mediate ADCC.
9. Mannose binding protein: A member of the collectin family of calcium-
dependent lectins, and an acute phase protein. It functions as a stimulator of
the classical pathway of complement activation, and as an opsonin for
phagocytosis by binding to mannose, a sugar residue usually found in an
exposed form only on the surface of microorganisms.
 10. Membrane attack complex (MAC): Complex of complement components
C5b-C9 which inserts as a pore into the membrane of target cells leading to
cell lysis.

FAQ:

1. What are the components of the membrane attack cpmplex in the

complement pathway
Ans: C5b, 6,7,8,9

2. What is anaphylotoxin?
Ans: Anaphylotoxin or complement peptides are fragments that are produced as
part of the activation of the complement system. It causes the release of histamine
and other mediators of immediate hypersensitivity from basophils and mast cells,
thereby producing sign and symptoms of immediate hypersensitivity without
involvement of IgE.

3. How the lectin pathway is get activated?

Ans: Lectin pathway is activated by the binding of mannose binding
lectin(MBL) to mannose residues on glycoproteins or carbohydrates on the
surface of microorganisms.

4. What is innate immunity?

Ans: Innate immunity refers to nonspecific defense mechanisms that come into
play immediately or within hours of an antigen’s appearance in the body.

Reference:

1. Ivan M. Roitt & Peter J. Delves; Roitt’s Essential Immunology; tenth

edition
2. Gabriel Virella; Medical Immunology; Fifth edition
3. Richard A. Goldsby, Thomas J. Kindt, Barbara A. Osborne, Janis Kuby;
Immunology; Fifth edition
Links:

1. http://www.complement-genetics.uni-mainz.de/
2. http://www.cehs.siu.edu/fix/medmicro/cfix.htm
3. http://www.gla.ac.uk/Acad/Immunology/compsyst.htm