Antigen Recognition Molecules pdf - D. Dhafer

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

Antigen recognition molecules

The recognition of foreign antigens is the hallmark of the specific adaptive immune
response. Two distinct types of the molecules are involved in these processes,
including immunoglobulins (Igs) and T-cell Ag-receptor (TCR).

Diversity and heterogeneity are characteristic features of these molecules. In both
cases, there is evidence of extensive gene-rearrangements which generate Igs or TCR
capable of recognizing many different Antigens.

A) Immunoglobulins (Ig):

Immunoglobulins,  also  known as  antibodies  (Ab)  are  group of glycoproteins  present
in the serum or tissue fluids of all mammals, including human. They are produced by
B-lymphocytes  when  the  host  in  contact  with  immunogenic  foreign  molecules
(antigens).  They  act  as  a  critical  part  of  the  immune  response  by  specifically
recognizing and binding to particular antigens, such as bacteria or viruses, and aiding
in their destruction.

Structure of Igs

Five distinct classes of Ig molecules are recognized, namely IgG, IgM, IgA, IgD and
IgE. Each molecule has two functional regions, a fragment of Ag-binding (Fab) and a
fragment crystallizable region  (Fc) mediate so  called effector's functions. Fab region
is a region of the antibody that binds to antigens. It is composed of one constant and
one  variable  domain  of  each  of  the  heavy  and  the  light  chain.  Fab  region  contains
variable sections that define the specific target that the antibody can bind. Fc region is
a  tail  region  of  an  antibody  that  interacts  with  cell  surface  receptors  called  Fc
receptors  and  some  proteins  of  the  complement  system  like  CIq.  Fc  region  of  all
antibodies  in  a  class  are  the  same  for  each  species;  they  are  constant  rather  than
variable.

The class and subclass of the Ig molecule are determined by its heavy chain type.
Thus, the four human IgG (1, 2, 3&4) have heavy chain called gamma1, 2, 3&4, the
two human IgA (1&2) have heavy chains called alpha 1&2. but none have been
described for IgM (mu-chain), IgD (delta-chain) or IgE (epsilon-chain).

All Igs are glycoproteins, but the carbohydrate content ranges from 2-3% for IgG, to
12-14% for IgM, IgD & IgE. IgG is the major Ig in normal human serum, accounting
for  70-75%  of  the  total  Ig-Pool,  followed  by  IgA  which  represents  15-20%  of  the
human  serum  Ig-pool,  then  IgM  which  accounts  for  approximately  10%  of  the  Ig-
pool,  then  IgD  which  counts  for  less  than  1%  of  the  total  plasma  Ig  but  is  a  major
component of the surface membrane of many B-cells. IgE, though scarce in serum, is
found on the surface membrane of basophiles and mast cells in all individuals.

The  basic  four  chain  model  for  Ig  molecule  is  based  on  two  distinct  types  of
polypeptide  chain.  The  smaller  (light)  chain  is  common  to  all  classes  and  show  to
exist in two distinct forms called kappa (κ) or lambda (λ), whereas, the larger (heavy)
chain which is structurally distinct for each class or subclass and called gamma (γ) in
IgG, alpha (α) in  IgA, Mu (μ) in  IgM, delta (δ) in  IgD, and epsilon (ε) in  IgE. Each
chain  consists  of  two  parts,  the  N-terminal  of  the  chain  shows  much  sequence

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

variability  and  known  as  variable  region  (V)  and  c-terminal  of  the  chain  which  is
constant  and  called  constant  region  (C).  The  constant  portion  of  the  heavy  chain  is
further divided into three structurally discrete regions:CH1, CH2, CH, these globular
regions  stabilized  by  intrachain  disulphide  bound  and  are  referred  to  as  “Domains”.
The  hinge  region  is  a  segment  of  heavy  chain  between  the  CH1  and  CH2-domains,
flexibility in this area permits the two Ag-binding sites to operate independently.

All light chains have one variable and one constant region. Whereas, gamma chain,
delta-chain ands alpha chain consist of three domains and the mu and epsilon chains
of IgM and IgA have an extra constant region domain. IgA and IgM have additional
peptide chain, the J (joining) chain, thought to assist the processes of polymerization.

If j-chains are not freely available, there is evidence that hexameric IgM becomes the
preferred form. Secretary IgA made up of two units of IgA, one secretary component
and one j-chain. Secretary component is not synthesized by plasma cells but by
epithelial cells. Bound secretary component facilitates the transport of S-IgA into
secretions, as well as protecting it from proteolytic attack.

Properties of Igs: The table below shows the properties of Igs

Properties

IgG

IgM

IgA

IgE

IgD

%in serum

0.004

0.2

M.W (X1000)

160

900

170-400

190

180

Half life

2.5

Molecular form

Monomer Pentamer Monomer

or dimer

Monomer Monomer

Subclasses

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

H chain

J chain

Secretory piece

Complement fixation

Crosses placenta

Mediates allergy

Opsonization

Binds to Fc receptor in
NK cells or phagocytes
(ADCC)

Ag receptor on B cells

Interaction of antibodies with antigens:

The binding of Ag to Ab involves the formation of multiple non-covalent bounds
between the Ag and amino acids of the binding site. The attractive forces (hydrogen
and electrostatic bounds, Van der Waals and hydrophobic forces) are weak by
comparison with covalent bounds. However, the large number of interaction results in
a large total binding energy.

The  strength  of  a  non-covalent  bond  is  critically  dependent  on  the  distance  between
the interacting groups and in order for epitope and an Ab-combing site (paratope) to
combine,  there  must  be  suitable  atomic  groupings  on  both  parts  (Ag  &  Ab)  and  the
shape of the combining site must fit the epitope. The steric repulsive forces come into
play  since  it  is  inversely  proportional  to  the  distance  between  the  electron  clouds.
These forces that determining the specificity of the Ab for a particular Ag.

The strength of the single bond between Ag and Ab is known as the Ab-affinity, it is
the sum of the attractive and repulsive forces, whereas the overall strength of
interaction between the Ab and Ag indicates Ab-avidity. The avidity is likely to be
more relevant than affinity, as naturally occurring Ags are multivalent. When some of
the epitopes of an Ag are shared by another Ag then a proportion of the Abs directed
to that Ag and this phenomenon is termed cross-reactivity.

Function of the antibodies:

Major functions of the antibodies are:

1. Neutralization of infectivity:

Antibodies  are  secreted  into  the  blood  and  mucosa,  where  they  can  block  the
infectivity  of  pathogens  (bacteria,  viruses,  parasites  and  fungi),  inactivate  or
neutralize foreign substances such toxins.  Neutralization generally occurs as a  result
of interfering with an organism’s attachment to host tissues.

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

some antibodies have been shown to  inhibit infectivity by binding to  organisms  and
causing  them  to  aggregate.  Aggregation  or  agglutination  by  IgA  may  allow  more
efficient  entrapment  of  bacteria  in  mucous  and  subsequent  clearance  by  peristalsis.
Although  aggregation  is  more  likely  to  occur  with  polymeric  IgA  and  IgM,  some
neutralizing  IgG  antibodies  can  aggregate  polio  virus  and  reduce  the  infectivity.
Similarly, Antibodies against HIV-1 gp120 interfere with binding of gp120 to CD4.

2. Phagocytosis:

Antibodies  facilitate  phagocytosis  of  foreign  substances  by  a  process  called
opsonization.  The  internalization  and  degradation  of  antibody-coated  pathogens  by
macrophages and neutrophils via FcRs (Fc receptors are protein molecules present on
the  surfaces  of  macrophages  and  neutrophils  which  can  bind  the  constant  region  of
immunoglobulin molecules) is a critical antibody function for clearance of pathogens
in vivo.

The  binding  of  phagocyte  Fc  receptors  with  several  antibody  molecules  complexed
with  the  same  target  initiates  a  signal  transduction  pathway  that  results  in  the
phagocytosis  of  the  antigen-antibody  complex.  Inside  the  phagocyte,  the  pathogen
becomes  the  target  of  various  destructive  processes  that  include  oxidative  damage,
enzymatic digestion, membrane disrupting effects of antibacterial peptides etc.

3. Antibody-dependent cellular cytotoxicity (ADCC):

Antibodies  have  shown  anti-microbial  activity  either  directly  or  through  interactions
with  FcRs  or complement.  ADCC occurs when  antibody forms a bridge  between  an
infected target cells (virus infected cells of the host) and an FcR-bearing effector cell,
particularly  natural  killer  (NK)  cells.  The  result  of  this  three-way  interaction  is  the
death of the target cell, either by lysis or apoptosis.

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

4. Complement-mediated lysis of pathogens or of infected cells: Antibodies

activate the complement system to destroy bacterial cells by lysis

Antibodies (IgM and most IgG subclasses) activate the complement system which can
result in the lysis of organisms or of infected cells. An important byproduct of the
complement cascade is C3b, which is a protein fragment that can bind nonspecifically
to cell and Ag-Ab complexes. Many cell types, for example, red blood cells or
macrophages have receptors for C3b and so bind cells or complexes to which C3b has
adhered.

Binding of Ag-Ab complexes by the C3b receptors of an RBC allows it to deliver the
complexes  to  liver  or  spleen  where  resident  macrophages  remove  them  without
destroying  red  blood  cell.  In  addition,  organisms  or  Ag-Ab  complexes  bound  by
complement  can  be  internalized  by  phagocytic  cells,  with  the  resultant  clearance.
Internalization through complement receptors on antigen-presenting cells (APCs) can
also result in the processing of antigen for presentation to T lymphocytes.

5. Transcytosis, mucosal immunity & neonatal immunity

Some antibodies can move across epithelial layers (depends on the property of the
constant region of that antibody molecule) via a process called transcytosis. IgA is the
major immunoglobulin that undergoes transcytosis and is available in secretory form
(sIgA) in the mucosal surfaces of respiratory, gastrointestinal and urogenital tracts.

In  mammalian  species  including  humans,  most  subclasses  of  IgG  can  cross  the
placental  barrier  (since  maternal  and  fetal  circulatory  system  are  separate)  thus
conferring  sample  of  mother’s  repertoire  of  antibody  to  the  developing  fetus  as
protective  endowment  against  pathogens.  This  passive  immunization  of  developing
fetus occurs during the third trimester of gestation.

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

Type of Antibody Major Function (s)

IgG

Opsonization, Complement Activation, Antibody dependent cell mediated

cytotoxicity, Neonatal immunity, Feedback inhibition of B cells

IgA

Mucosal Immunity

IgM

Naive B cell antigen receptor, complement activation

IgD

Naïve B cell receptor

IgE

Defense against helminthic parasites, immediate hypersensitivity

diversity:

Generation of Ab

Abs are remarkably diverse and provide enough different combining sites to
recognize the millions of antigenic shapes in the environment. In fact, we produce
more types of Ab than there are genes in our genome. All these diversities generated
by:

• Multiple V-region genes in the germ line; i.e many gene coding for the

variable (V) region.

• Somatic recombination between elements forming a V-region gene. i.e a

number of gene segments could recombine to give a complete V-gene.

• Gene conversion; a panel of pseudo genes can also be used to alter the

sequences within the variable region.

• Nucleotide addition; during cutting and joining of the DNA extra nucleotides

may be inserted.

• Somatic mutation; so the relatively few genes give rise to many mutated genes

during the lifetime of the individual.

Mammals may use all five mechanisms to generate diversity and another

possible  mechanism  like  recombinational  inaccuracies  and  assorted  heavy  and  light
chain.  In  humans  the  genes  encoding  kappa  and  lambda  light  chains  are  found  on
chromosomes 2 and 22 respectively whereas, the heavy chain gene locus is found on
chromosome 14. the genes for isotypic variants are gamma 1,2,3&4 , mu, alpha1&2,
delta, epsilon, kabba and lambda-chain genes, all present in human geneome (Isotypic
varation).  Thus,  isotypic  variation  is  present  in  the  germ  line  of  all  members  of  a
species,  producing  the  heavy  and  light  chains  and  the  V-region  framework
(Subgroups).  Allotypic  variation  is  interspecies  allelic  variability.  Allotypes  occur
mostly as variants of heavy chain constant regions. It is not found in all people and is
therefore an allotype.

The idiotypic variation refers to the diversity at the Ag-binding site (paratope)

and in particular relates to the hyper variable segments. These determine the binding
specificity of the Ag-binding site. The hyper variable regions are sometimes referred
to as complementarity determining region (CDRs).

Dr Dhafer A. Alghezi

Lecture 4: Antigen recognition molecules

Immunoglobulin gene recombination:

Light chain genes recombine V and J segments to make a gene for the VL

domain. Whereas, heavy chain genes recombine V, D and J gene segments to make a
gene for the VH domain.
Several Vk-genes on the germ line DNA (V1-Vn) and Jk segment (Jk1-Jk5) and only
one constant-region (k-gene). But in the lambda-gene a set of V-genes (v1-Vn), just
one J-gene and seven C-genes. Rearrangement of the V and J genes recombined with
–region to form light chain.

Also in the heavy chain region the variable region encoded by V and J

segments genes and a third gene segment, the D (diversity)-segment gene. The D is
highly variable both in number and sequence of base paired.

VH (V1-Vn), DH (D1-D30), J (J1-J6). More than one D-segment may join to

form an enlarged D-region. Eighty seven VH segments are found on chromosome 14,
of which at least 32 are pseudo genes. The recombination of V,D,J segments in the
heavy chain is largely responsible for the variability of CDRs. The place at which V
& J segment gene join may vary slightly and these slight variations in the position of
recombination generate additional diversity.
Somatic mutation:-

The Ig heavy and light chain genes undergo structural modifications after Ag-

stimulation. There is some evidence that V-region is particularly mutable. The
somatic mutation occurs in germinal centers to produce a higher-affinity Ab. This
process called affinity maturation. So the potential for increased diversity multiplies at
each step of Ig-production.

2. T cell antigen Receptor (TCR):