Amino acids & Proteins

Antibody Diversity
• The human genome contains less than 150
immunoglobulin genes.
• Nevertheless, each person is capable of
synthesizing perhaps 1 million different
antibodies, each specific for a unique antigen.
 Antibody Diversity
• Immunoglobulin diversity is generated by
combinatorial mechanisms based upon
mixing and rearranging a finite pool of genetic
information in multiple ways.
• The first source of antibody diversity is the
division of the coding sequence for each
immunoglobulin chain among multiple genes.
Genes of Light chains of antibodies
• Each immunoglobulin light chain is the
product of at least three separate structural
genes:
1. A variable region (VL) gene,
2. A joining region (J) gene
3. A constant region (CL) gene.
 Genes of Heavy chains of antibodies
• Each heavy chain is the product of at least
four different genes:
1. A variable region (VH) gene,
2. Diversity region (D) gene,
3. A joining region (J) gene, and
4. A constant region (CH) gene.
 Antibody Diversity
• Diversity is further augmented through the
action of the activation-induced cytidine
deaminase (AID).
• By catalyzing the conversion of cytidine to
uracil, AID massively increases the frequency
of mutation of immunoglobulin V genes.
• These mutations are somatic in nature, ie,
unique to a differentiated cell rather than to a
germline cell.
 Antibody Diversity
• Consequently, each activation of AID generates
new subpopulations of B cells that harbor unique
mutations of their V genes, causing each to
synthesize immunoglobulins of differing antigen
specificity.
• In some pathologic states, the mutagenic action of
AID can lead to the generation of autoantibodies
that target the body’s endogenous components, a
phenomenon termed autoimmunity.
 Antibody Diversity
• A third mechanism for generating antibodies
targeting novel antigens is junctional diversity.
• This refers to the addition or deletion of random
numbers of nucleotides that takes place when
certain gene segments are joined together.
• As is the case with AID, the mutations generated
by junctional diversity are somatic in nature.
 Class (Isotype) Switching
• The transition from the synthesis of one class
to another is designated class or isotype
switching.
• Each class appears in a specific chronologic
order in response to the immunogen
(immunizing antigen).
• For instance, antibodies of the IgM class
normally precede molecules of the IgG class.
• Switching involves the combining of a given
immunoglobulin light chain with different
heavy chains.
• A newly synthesized light chain will initially be
mated with a μ chain to generate a specific
IgM molecule.
• Over time the same antigen specific light chain
will be mated with a γ chain.
• The γ chain will, however, possess an identical
VH region to that of the μ chain, thus
generating an IgG whose antigen specificity is
identical to that of the original IgM molecule.
• The same light chain can also combine with an
α heavy chain, again containing the identical
VH region, to form an IgA molecule with
identical antigen specificity.
• Immunoglobulin molecules of different classes
that possess identical variable domains and
antigen specificity are said to share an
idiotype.
• Idiotypes are the antigenic determinants
formed by the specific amino acids in the
hypervariable regions.
 Complement system

• Activated by invading organisms &, more

often, triggered by antibodies ('complements'
action of antibodies).
• Consists of some 20 plasma proteins produced
by liver.
• The complement system is named because it
"complements" the white blood cells' forces. it
is involved in the MAC attack, which stands for
"Membrane Attack Complex.
• The complement system displays features
reminiscent of the blood’s coagulation cascade.
• Both consist of sets of circulating zymogens
(proproteins) that remain catalytically dormant
until activated by proteolytic cleavage.
• These proteins, called complement factors, are
synthesized by a variety of cell types, including
hepatocytes, macrophages, monocytes, and
intestinal endothelial cells.
• As is the case for clotting factors, most
complement factors are proproteases that,
upon activation, target other components of
the system, thereby generating a series or
cascade of proteolytic activation events that
amplify the production of the system’s
protective end products.
 Classical Complement pathway
• The classical pathway for activating the
complex system is triggered when an
antibody-antigen complex binds to and
stimulates the protease activity of factor C1.
• C1 then cleaves complement factor C2 to form
two smaller proteins, C2a and C2b, and
cleaves complement factor C4 to form C4a
and C4b.
• The C5b protein then combines with
complement factors C6, C7, C8, and C9 to
form the membrane attack complex (MAC).
• MAC kills bacterial invaders by binding to and
opening a pore in their plasma membrane.
• Following lysis, the bacterial remains are
destroyed by phagocytic macrophages.
• Meanwhile, the C3a and C5a proteins serve as
chemoattractants that recruit leukocytes to
the site of infection and stimulate an
inflammatory response.
• Targeting of the MAC to invading bacteria is
facilitated by the presence of thioester bonds
in C3 and C4.
• Like the thioester bond in the plasma protease
inhibitor α2-macroglobulin, this highly reactive
bond becomes exposed as a result of the
conformational change that accompanies
activation.
• In the case of C3 and C4, the thioester reacts with
the hydroxyl groups of the bacteria’s surface
polysaccharides, covalently anchoring them and
the C5 convertase complexes of which they are a
part to their target pathogen.
• Consequently, the components of the MAC
are formed and assemble together in close
proximity to the bacterial membrane.
Compliment pathways
 Lectin Complement pathway
• Activation can also be triggered via the lectin
pathway, wherein the complexes formed
when a complement factor known as
mannose-binding lectin (MBL), also known as
mannan-binding protein (MBP), binds
bacterial polysaccharides to generate a
complex that recruits and activates C4.
• The term lectin refers to any protein that
binds polysaccharides.
• Most lectins are highly selective.
• MBL is specific for the mannose-containing
carbohydrate moieties (mannans) of
glycoproteins and lipopolysaccharides present
on the surface of Gram-positive bacteria,
some viruses, and several fungi.
• Upon binding to the polysaccharide-MBL
complex, C4 undergoes autoproteolysis,
forming C4a and C4b, In addition, it cleaves C2
into C2a and C2b.
• The activation cascade then proceeds in the
same manner as described for the classical
pathway.
Compliment pathways
• MBL circulates as large, multivalent complexes
consisting of four or more copies of a
homotrimeric core unit made up of of three
copies of a ≈ 30 kDa polypeptide.
• Each polypeptide contains two major domains:
 Amino-terminal collagen-like domain
 A carboxyl-terminal carbohydrate-binding
domain.
• The core of the homotrimer is formed when three
collagen-like domains intertwine to generate an
extended tail that leads to a globular head
consisting of the three carbohydrate-binding
domains.
• The homotrimers associate via their amino terminal
tail regions into a disulfide linked “stalk” from
which the individual carbohydrate binding heads
extend in a branched arrangement resembling that
of the immunoglobulins
Mannose binding lectin
 Alternative Complement pathway
• The complement system also can be activated
by the alternative pathway, which involves the
activation of C3 by chemical hydrolysis, a
process sometimes referred to as “ticking over.”
• In the alternative pathway, C3b binds
complement factor B, forming a C3b:B complex
that is then cleaved by complex factor D. The
resulting C3b:Bb complex possesses C
convertase activity.
Compliment pathways
 Major functions of
Immunoglobulins

Immunoglobulin G:

• Main antibody in the secondary response.

1. Opsonizes bacteria, making them easier to
phagocytose. (Antibody opsonization is the process by
which a pathogen is marked/labelled/identified for
ingestion/destruction and eliminated by a phagocyte.)
2. Fixes complement, which enhances bacterial killing.
3. Neutralizes bacterial toxins and viruses.
4. Crosses the placenta.
• Immunoglobulin A:
1. Secretory IgA prevents attachment of
bacteria and viruses to mucous membranes.
2. Present in the external secretions like milk,
tears, saliva, bronchial and intestinal
secretions.
3. Does not fix complement.
• Immunoglobulin M:
1. It is the first antibody to appear in the serum
when an antigen is injected- primary antibody
response, then followed by IgG
2. Produced in the primary response to an
antigen.
3. Activates complement.
4. Does not cross the placenta.
5. Important Ig in autoimmune diseases.
• Immunoglobulin D:
Found on the surfaces of B cells where it acts
as a receptor for antigen.
• Immunoglobulin E:
1. Mediates immediate hypersensitivity by causing
release of mediators from mast cells and
basophils upon exposure to antigen (allergen).
2. Defends against worm infections by causing
release of enzymes from eosinophils.
3. Does not fix complement.
4. Main host defense against helminthic infections.
• Bence Jones proteins:
• Not a normal protein.
• Found in the plasma of multiple myeloma
patients, in whom large amounts of one type of
antibody is produced.
• Light chains of Igs.
• It is coagulated at 50-60 degrees, but on further
heating it dissolves.
• Multiple myeloma- in urine of such patients.
 Summary
Our bodies can synthesize immunoglobulins specific
for as many as a million different targets, called
antigens.
■ The core structure of the immunoglobulins is a
tetramer consisting of two light and two heavy
chains arranged in a “Y” configuration.
■ Synthesis of diverse antibodies from a limited set
of genes is made possible by combining,
rearranging, and somatic mutation of
immunoglobulin genes.
 Summary
■ The complement system is generally activated by
complexes formed between infecting microbes
and protective antibodies
■ The complement system is activated by a series of
proteolytic cleavage events that transform
dormant zymogens into active proteases.
■ Autoimmune disorders result when the immune
system attacks our body’s own tissues