Antibody Structure: Investigating Antibody Structure Igg Molecule Recap Important Areas

Antibody Structure
Investigating Antibody Structure
IgG Molecule Recap
Important areas:
Amino terminus
Carboxyl terminus
Hinge region
Characteristics:
Bivalent
Heterodimer
Disulfide bonded
Globular like structure
Hinge region allows movement
(Proline)
Antibody Functional Parts
Structure of Ig was known
Investigation of functional groups

What parts of the Ig did what?
Proteases used to digest below disulphide bond on
heavy chain
Pepsin
Papain
F(ab)2 still bivalent, therefore can bind 2 sites

Therefore can still act like and antibody
If reducing agent used, forms monovalent Ab

structure---no globular structure can form
Note:
FC region is responsible for secondary biological

function
Ex.
Ability to cross the placenta
Ability to fix compliment
Antibody Diversity
We know that there is a high level of diversity in the IRS; where
does
this come from?
How do we get specificity region and biological function

incorporated
into Ab?
Initial studies
• Dr. Hood injected Balb c mice with multiple myeloma and
precipitated out the light chains of IgG
• Found out that although mice were genetically identical, the IgG
samples from the mice were not.
• After sequencing, found that variations lay in specific area.
• Hood suggested that 2 genes encode for a single polypeptide chain
giving rise to a variable and constant region
• Notice that there is a high mutation rate in the V region
• Therefore variability is created allowing a response to a wide range
of antigens.
Antibody Diversity
How do you make a variable and constant region on the same molecule?
Dreyer and Bennet Proposed the 2-gene Model
• Suggested that two separate genes encode a single Ig heavy or light chain.
One gene for the the V region and the other for the C region.
• Proposed that these two genes come together at the DNA level to form
continuous message that can be transcribed and translated into a single Ig
heavy or light chain.
• Proposed that multiple V region genes were carried in the germ

line, but only single copies of C region genes exist.
• By postulating a single C region gene for each Ig class and subclass, the
model
could also account for the conservation of necessary biological effector
functions while allowing for evolutionary diversification of V-region genes.
Antibody Diversity
• Further Studies of Antibodies
• Since there is such a diverse pool of Ig’s in
the body, the best way to study them is by
isolating the H chain
• You wouldn’t isolate out the light chains as
all Ig ‘s share light chain types (kappa and
lambda).
• The heavy chain is what sets them all
apart.
Ig Heavy Chains
Separation of heavy chains
• Separation column used
• Heavy chains of particular classes will elute at different MW
• In constant region of heavy chain there are 5 isotypes (µ,δ,γ,ε,α)
• The heavy chains of the antibodies determine what class it is (ie IgA= α)
• Each class can have either k or λ light chains
Make-up of heavy and light chains
k or λ light chain families contain V J C gene segments

Rearranged V J segments encode the variable region of the light chain.
The heavy chain family contains V D J C genes segments

Rearranged V D J segments encode the variable region of the heavy chain.
In Humans k = 70% In Mice k = 95%

λ = 30% λ = 5%
Ig Heavy Chains
Variation within C region of heavy chain
Classes Subclass Light Chain

IgA α α1 α2 κλ
IgD δ δ κλ
IgG γ γ1 γ2 γ3 γ4 κλ
IgE ε ε κλ
IgM µ µ κλ
• How many different genes are responsible for coding for Ig’s?
__different genes encoding for Ig’s = diversity
• What part of the Ab molecule does the 20 Ab recognize?
• How many sub forms of IgG are there?

Ig Diversity
How did these subtle differences arise?
cys cys
V
C1
C2
Since common elements are seen, there is a lot of homology
Indication that there is a common ancestor

Ab Diversity
Definition of a subclass.
Hemoglobin (Hb)
Over our lifetime there are 4 types of Hb
I. Embryonic – α2ε2 γ2 ε2 α1
α2
II. Fetal -- α2 γ2 δγ2
1
III. Adult – major α2 β2 β2
IV. Adult – minor α2 δ2
• 5 genes involved here

Gene duplication
• High homology
Ab Subclasses
Phylogenetic tree based on
β
ε α
•Size δ
•Function
•Homology γ
Events that will give rise to diversity
•Gene duplication
•Inversion
•Translocation
•Deletion
Where do the differences exist on
the Antibody?
1. Isotypic determinants are

constant region determinants that
distinguish each Ig class and
subclass within a species.
2. Allotypic determinants are subtle

amino acid differences encoded
by different alleles of isotype
genes.
Allotypic differences can be
determined by comparing the
same Ab class among
inbred strains
3. Idiotypic determinants are

generated by the conformation of
the amino acid sequences of the L
and H chain V regions. Each
determinant = idiotope. The sum
of these = idiotype.
There are two types of allotypic differences:
1. Simple Allotypic differences

Ex k light chain
k1
k2
C region
Single amino acid difference @ 1 point
2. Complex Allotypic differences

ex 2 alleles that are different by 30%
What is the mechanism by with alleles can differ by
30%?
Events that will give rise to diversity
Unequal Crossing Over
Haptoglobin
Binds to Hb and scavenges Fe
Hp1 --- 83 aa
Hp2 --- 142aa
Primates only have Hp1

Humans have both Hp1 and Hp2
How could this have developed?
α1 83aa Hp1
α2 142aa Hp2
sequence starts at 12 and repeats

Mechanism Expansion and
α1 83aa Hp1 contraction of
71aa
gene families
α1 83aa
12aa
α2 142aa Hp2
Misalignment and crossing within the gene gives rise to α2

α2 is a better scavenger of Fe (evolution)

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Antibody Structure

Investigating Antibody Structure

IgG Molecule Recap

Investigation of functional groups

F(ab)2 still bivalent, therefore can bind 2 sites

If reducing agent used, forms monovalent Ab

FC region is responsible for secondary biological

How do we get specificity region and biological function

Dreyer and Bennet Proposed the 2-gene Model

• Proposed that multiple V region genes were carried in the germ

• Heavy chains of particular classes will elute at different MW

• In constant region of heavy chain there are 5 isotypes (µ,δ,γ,ε,α)

• Each class can have either k or λ light chains

Make-up of heavy and light chains

k or λ light chain families contain V J C gene segments

The heavy chain family contains V D J C genes segments

In Humans k = 70% In Mice k = 95%

Classes Subclass Light Chain

• What part of the Ab molecule does the 20 Ab recognize?

• How many sub forms of IgG are there?

How did these subtle differences arise?

Since common elements are seen, there is a lot of homology

Indication that there is a common ancestor

• 5 genes involved here

Events that will give rise to diversity

1. Isotypic determinants are

2. Allotypic determinants are subtle

3. Idiotypic determinants are

1. Simple Allotypic differences

2. Complex Allotypic differences

Binds to Hb and scavenges Fe

Primates only have Hp1

How could this have developed?

sequence starts at 12 and repeats

Misalignment and crossing within the gene gives rise to α2

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