Structural Biology and

Functions
of Immunoglobulin's

Organization and Expression of

Immunoglobulin Genes
Kasi.M.Kannan
Lecturer in Microbiology
V.H.N.S.N.College
Virudhunagar
 Antibodies: Structure and
Function
• Antibodies are the antigen binding proteins
present on the B-cell membrane and secreted
by plasma cells.
• Membrane-bound antibody confers antigenic
specificity on B cells; antigen-specific
proliferation of B-cell clones is elicted by the
interaction of membrane antibody with
antigen.
• Secreted antibodies circulate in the blood,
where they serve as the effectors of humoral
immunity by searching out and neutralizing
antigens or marking them for elimination.
• All antibodies share structural features, bind
to antigen, and participate in a limited
 Basic Structure of Antibodies
• The first evidence that antibodies were
contained in particular serum protein
fractions came from a classic experiment by
A. Tiselius and E. A.Kabat, in 1939.

• They immunized rabbits with the protein

ovalbumin (the albumin of egg whites) and
then divided the immunized rabbits’ serum
into two aliquots.

• Electrophoresis of one serum aliquot revealed

four peaks corresponding to albumin and the
• The other serum aliquot was reacted
with ovalbumin, and the precipitate that
formed was removed; the remaining
serum proteins, which did not react with
the antigen, were then electrophoresed.
• A comparison of the electrophoretic
profiles of these two serum aliquots
revealed that there was a significant
drop in the γ –globulin peak in the
aliquot that had been reacted with
antigen
• Thus, the globulin fraction was identified
as containing serum antibodies, which
were called immunoglobulins, to
distinguish them from any other proteins
that might be contained in the γ
-globulin fraction.
• Experimental
demonstration that most
antibodies are in the γ
-globulin fraction of serum
proteins.
• After rabbits were
immunized with ovalbumin
(OVA), their antisera were
pooled and
electrophoresed, which
separated the serum
proteins according to their
electric charge and mass.
• The blue line shows the
electrophoretic pattern of
untreated antiserum. The
black line shows the pattern
of antiserum that was
incubated with OVA to
remove anti-OVA antibody
and then electrophoresed
 Structure of Immunoglobins

• IgG Y shaped
• Antigen binding site located on
tip of the Y arms
• Fab arms connected to Fc stem
domain via a flexible hinge
• Two identical H chains and L
chains
• Each chain has a N-terminal VH
and VL domain that together form
the antigen binding site
• Covalent disulfide bridges
between H and H-L
Structure of antibody
Antibody Structure
Antibody Structure
 Constant and Variable regions of
• C Antibodies
terminus of both H and L chains
• invariant and defined as C
(constant) region.
• Length ~ 330 amino acids in H
chains and ~ 110 amino acids in L
chains

• N terminal segment
• Substantially different in different
antibodies, thus named V
(variable) region

• V region ~ 110 amino acids in

length
• Contains three subregions that
show maximal variation between
different antibodies
• Defined as hypervariable regions
• Designated as complementarity
determining regions (CDRs)
The Immunoglobin Fold
 Immunoglobin Fold
• V and C domains share the basic
Ig fold

• Differences between the two

domains

• C domain is built of seven β

-strands arranged so that four
strands form one sheet and three
strands form a second sheet.

• The strands are closely packed

together and joined by a single
disulphide bond

• Most of the invariant residues of

the constant domain are in the
sheets

• Overall structure of the V domain

Variable and
Constant Folds
Domain Structure of Immunoglobulin's
Domains are folded, compact, protease resistant structures

Fc Fab

S Light chain C
S
VL domains
C L

S S
S S
3
H
C

CH1
CH2
S VH
S
Heavy chain C
domains
F(ab)2

Pepsin cleavage sites - 1 x (Fab)2 & 1 x Fc

Papain cleavage sites - 2 x Fab 1 x Fc
CH3
CH2

CH3
 CH1

CH2

CH3
VH1
CH1

CH2

CH3
VH1
CH1

VL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
VH1
CH1

VL CL

CH2

CH3
 VH1
CH1

CL VL

CH2
Elbow

Hinge

CH3
Flexibility and

Fv
motion of

Fv
Fv
immunoglobuli Elbow
ns

Fb

Fb
Fv
Fb Fv
Hinge
CH2

CH2
3

3
H

H
C

C
 Fv

VH1
CH1 Fb

CL VL

Fab

CH2
Elbow

Hinge
Fc
Carbohydrate

CH3
 The Immunoglobulin Fold
The characteristic structural motif of all Ig domains

A β barrel of 7 (CL) or 8 (VL)

polypeptide strands connected A barrel made of a sheet of
by loops and arranged to staves arranged in a folded
enclose a hydrophobic interior over sheet

Single VL domain Barrel under construction

 The Immunoglobulin Fold

COOH

S S

NH2

Unfolded VL region showing 8 antiparallelβ -pleated

sheets connected by loops.
mmunoglobulins are Bifunctional Proteins

•Immunoglobulins must interact with a finite

number of specialised molecules -
Easily explained by a common Fc region irrespective of specificity

•- whilst simultaneously recognising an infinite

array of antigenic determinants.

In immunoglobulins, what is the structural basis

for the infinite diversity needed to match the
antigenic universe?
ariability of amino acids in related proteins
Wu & Kabat 1970
100
Variability
80

60 Cytochromes C
40

20

20 40 60 80 100 120

Amino acid No.

100
Variability
80 Human
60 Ig heavy
chains
40

20

20 40 60 80 100 120

Amino acid No.

Framework and Hypervariable regions
•Distinct regions of high variability and conservation led to the
concept of a FRAMEWORK (FR), on which hyper variable regions were
suspended.

•Most hypervariable regions coincided with antigen contact points

- the COMPLEMENTARITY DETERMINING REGIONS (CDRs)

FR1 CDR1 FR2CDR2 FR3 CDR3 FR4

100
Variability
80

60

40

20

20 40 60 80 100 120

Amino acid No.

Hypervariable CDRs are located
on loops at the end of the Fv regions

Hypervariable regions
Space-filling model of (Fab)2, viewed from above,
illustrating the surface location of CDR loops

Light chains Green and

brown
Heavy chains Cyan and blue
Hypervariable loops and
framework:
Summary
•The framework supports the hyper variable loops it
forms a compact β barrel/sandwich with a
hydrophobic core
•The hypervariable loops join, and are more flexible
than, the β strands
•The sequences of the hypervariable loops are
highly variable amongst antibodies of different
specificities
•The variable sequences of the hypervariable loops
influences the shape, hydrophobicity and charge at
the tip of the antibody
Antigens vary in size and complexity

Protein:
Hapten:
Influenza haemagglutinin
5-(para-nitrophenyl
phosphonate)-pentanoic acid.
 Antibodies interact
with antigens in a
variety of ways

Antigen inserts into a

pocket in the antibody

Antigen
interacts with
an extended
antibody surface
or a groove in
the antibody
surface
he Complementarity Determining Region
• Six loops of the VH (H1, H2 and H3) and VL (L1,
L2 and L3) domains create a great variety of
surfaces

• Deep binding cavities: such as those seen in

some antibody-hapten complexes

• Wide pockets : seen in certain antibody-peptide

complexes

• Flat surfaces : seen in antibody-protein

interactions

• H3 is the most variable of the loops and in all

crystallographically solved antibody-antigen
What Do Antibodies Recognize?

• Proteins (conformational determinants,

denatured or proteolyzed determinants)
• Nucleic acids
• Polysaccharides
• Some lipids
• Small chemicals (haptens)
Antigen:Antibody
complex
• Antibodies bind to
antigens by recognizing a
large surface, and through
surface complementarity.

• Thus, these complexes

have a very high affinity
for each other.
Ig gene Super family - IgSF

• The genes encoding Ig domains are not

restricted to Ig genes.
• Although first discovered in
immunoglobulins, they are found in a
superfamily of related genes, particularly
those encoding proteins crucial to cell-
cell interactions and molecular
recognition systems.
• IgSF molecules are found in most cell
• Large numbers of membrane proteins have
been shown to possess one or more regions
homologous to an Ig domain.
• Each of these membrane proteins is
classified as a member of the
immunoglobulin superfamily.
• The term superfamily is used to denote
proteins whose corresponding genes derived
from a common primordial gene encoding
the basic domain structure.
• These genes have evolved independently
and do not share genetic linkage or function.
• The following proteins, in addition to the Ig
themselves, are representative members of
the immunoglobulin superfamily
• Ig-/Ig- heterodimer, part of the B-cell receptor
• Poly-Ig receptor, which contributes the
secretory component to secretory IgA and IgM
• T-cell receptor
• T-cell accessory proteins, including CD2, CD4,
CD8, CD28, and the , , and chains of CD3
• Class I and class II MHC molecules
• Beta 2-microglobulin, an invariant protein
associated with class I MHC molecules
• Various cell-adhesion molecules, including
VCAM-1, ICAM-1, ICAM-2, and LFA-3
• Platelet-derived growth factor
Numerous other proteins, also belong to the
immunoglobulin superfamily.
 Expression of Immunoglobulin
Genes
One of the most remarkable features of the
vertebrate immune system is its ability to respond to
an apparently limitless array of foreign antigens.
As immunoglobulin (Ig) sequence data
accumulated, virtually every antibody molecule
studied was found to contain a unique amino acid
sequence in its variable region but only one of a
limited number of invariant sequences in its constant
region.
The genetic basis for this combination of
constancy and tremendous variation in a single
protein molecule lies in the organization of the
immunoglobulin genes.
 Generation of antibody diversity:
at gene, antibody, cell and organism level

Heavy chain variable region has 3 segments: VH, DH, JH

Light chain variable region has 2 segments: VH, JH

1. Combinatorial joining
– Utilize any of the multiple gene segments
– Most 3’ is used most frequently
– Variable gene segment selection
Typical gene coding transmembrane protein
Separate exons code for individual domains
of Ig protein molecule
Genetics of Ig kappa light chain synthesis
DNA rearrangement for kappa light chain
”Looping out” unwanted genes
Construction
of Ig light
chain
Construction
of Ig heavy
chain
Genetics of Ig heavy chain synthesis
2. Junctional diversity

– Variable nucleotide lengths of J region

3. Hypermutation (somatic)

– Untemplated point mutations

– Hot spots of mutation in binding region
 Somatic hypermutation
(yields variation into rearranged Ig that is subject to
pos & neg selection to yield improved antigen binding)
4. Somatic gene conversion (few species)

– Translocation of short segments of V region

(pseudogenes) to VH gene
– Used when species has few VH genes or little
variation
– Chicken, rabbit
Somatic gene conversion (eg., chicken IgH)
Pseudogene sequences copied into rearranged B-cell VDJ unit
 Process of
gene
conversion:

pseudo genes
generate
sequence
diversity
Diversification
of chicken Ig
occurs
through gene
conversion
5. Different pairing of VH and VL chain

– To form antigen-binding site

– Combinatorial joining of chains (not gene
segments)
– Mechanism acting at the protein level
V-region genes constructed from gene segments
6. Class switching of immunoglobulin

– In specific cytokine environment

– VDJ is juxtaposed with different CH genes
– Same antigen-specificity, new function
– Occurs by removal of intervening DNA
– Structural variation in C regions confers
functional specialization
Class switching in Ig synthesis
Mechanism of
class
switching
 Isotype
switching
involves
recombin-
ation
between
specific
switch
signals
Co-expression of IgD and IgM
regulated by RNA processing
Different types
of variation
between
immunoglobulins
Differences in inheritance among
major immunoglobulin variants
Ig classes and subclasses in mammals
 Synthesis, Assembly, and
Secretion of Immunoglobulins
• The heavy and light chains are synthesized on
separate polyribosomes (polysomes).
• The assembly of the chains to form the
disulfide-linked immunoglobulin molecule
occurs as the chains pass through the cisternae
of the rough endoplasmic reticulum (RER) into
the Golgi apparatus and then into secretory
vesicles.
• The main figure depicts assembly of a secreted
antibody.
• The inset depicts a membrane-bound antibody,
which contains the carboxyl-terminal
transmembrane segment.
• This form becomes anchored in the membrane
of secretory vesicles and then is inserted into
the cell membrane when the vesicles fuse with
Synthesis, Assembly, and Secretion of
Immunoglobulins
• Transcription of immunoglobulin genes
is regulated by three types of DNA
regulatory sequences: promoters,
enhancers and silencers.
• Growing knowledge of the molecular
biology of immunoglobulin genes has
made it possible to engineer
antibodies for research and therapy.
• The approaches include chimeric
antibodies, bacteriophage-based
combinatorial libraries of Ig-genes, and
the transplantation of extensive