Download Genetics in the Generation of Antibody Diversity

Genetics in the Generation of
Antibody Diversity
Folder Title: AbGenes(NoTP)
Updated: October 2, 2016
Chapter Seven, 7th Edition:
“Organization and Expression of Lymphocyte Receptor Genes”
Includes Immunoglobulin and T-Cell Receptor Genetics
Questions About B-Cell Responses
Biochemical Questions:
(Slides 3 to 8 Review: Fab Variable Region Sequences)
1. How can Immunoglobulins recognize so many different
epitopes?
2. What does an antibody protein look like?
3. How do specific antibodies work as proteins binding so many
different specific antigenic determinants?
Representation of Sequence Comparisons Among Light Chains
from Antibodies with Three Different Antigen Specificities
H3N-Ser-Val-Ile-Thr-Gly-Gly-Tyr-Ala... Thr-Glu-Ala-Val-Tyr-Ser-Met-COOH3N-Ser-Ile-Met-Thr-Arg-Leu-Tyr-Gly..Thr-Glu-Ala-Val-Tyr-Ser-Met-COOH3N-Thr-Gly-Gly-Thr-Lys-Leu-Tyr-Ile..Thr-Glu-Ala-Val-Tyr-Ser-Met-COO-
Variable Amino Terminal Half
Conserved Carboxyl Terminal Half
(Positions 1 to 107)
(Positions 108 - 214)
LiteComp
See Figure 4-6,
Kuby 6th Edition
p. 85
Variable Regions
Including
hypervariable CDR’s
(Complementarity
determining regions)
κ or λ forms
See Figure 4-6,
Kuby 6th Edition
p. 85
Heavy Chain
Iso-forms
The Heavy and light chains are labeled incorrectly in the Kuby Immunology Powerpoint slides.
The figure is labeled correctly in the book.
Questions About B-Cell Responses
1.
Biochemical Questions:
How can Immunoglobulins recognize so many different epitopes?
2.
3.
What does an antibody protein look like?
How do specific antibodies work as proteins binding so many different specific
antigenic determinants?
Genetic Questions:
How can this diversity of structure leading to enormous diversity
of function be coded and controlled by a very limited host
genome?
How can we have “one gene - one polypeptide” and make a
virtually limitless selection of polypeptides?
Problems for Genetics in Generating
Antibody Sequence Diversity
Vast Sequence Diversity- Encoded by Very Limit Genome
Heavy and Light Chain Sequence VariationsAlmost Exclusively in the Amino Terminal One Half of
the Light Chains and the Amino Terminal One fourth
of the Heavy Chains
Exactly the same V-region sequencesEnd up on different C-region Isotypes.
How is all of this possible?????
Differentiated Neoplastic
Plasma Cell making a single
antibody.
Myeloma Cell DNA
Myeloma Cell Conclusions
Variable and conserved
regions of light chain are
linked in the differentiated
end-product cell line
DNA coding for the mRNA for
the light chain is all in one
Continuous sequence as for
any gene for any protein.
Embryonic Mouse Cell DNA
Variable and conserved
regions of light chain are
not linked originally in the
stem cell lineage!
Immunology and Phone Numbers
315-443-1870
212-345-1775
478-367-8903
537-503-2078
409-159-6309
610-970-3970
934-620-8122
909-603-7023
800-620-6021
704-590-5307
703-725-0153
207-502-6671
435-431-0890
412-830-0048
740-592-1954
307-620-4450
490-501-5672
601-909-7002
554-891-7712
335-592-0944
20 Phone Numbers
Immunology and Phone Numbers
315212478537409610934909800704703207435412740307490601554335-
-443-1870
-345-1775
-367-8903
-503-2078
-159-6309
-970-3970
-620-8122
-603-7023
-620-6021
-590-5307
-725-0153
-502-6671
-431-0890
-830-0048
-592-1954
-620-4450
-501-5672
-909-7002
-891-7712
-592-0944
400 Phone
Numbers
Immunology and Phone Numbers
315212478537409610934909800704703207435412740307490601554335-
-443-345-367-503-159-970-620-603-620-590-725-502-431-830-592-620-501-909-891-592-
-1870
-1775
-8903
-2078
-6309
-3970
-8122
-7023
-6021
-5307
-0153
-6671
-0890
-0048
-1954
-4450
-5672
-7002
-7712
-0944
8000 Phone
Numbers
41 Kappa V-Region Sequences in Humans
“Pseudo-gene” is not expressed
Kappa Chain DNA: Vk’s
This and the next two slides deal with Kappa Light Chain
DNA. Human Kappa DNA was 41 different “V” region genes
to work with, and 4 “J” region genes. (164 Possibilities)
Lambda light chain DNA works the same way except that the
Human Lambda light chain works with 33 “V” regions and 5
“J” region genes. (165 Possibilities)
Kappa Chain: K-J- C
Complete Kappa Chain
Light chain V-region genes do not have diversification genes
Heavy Chain Dna: VH’s, CGenes
This and the next two slides deal with Heavy chain DNA. Human
heavy chain DNA has 41 “V” region genes to work with, 23 “D”
(diversification) region genes, and 6 “J” (joining) region genes. Heavy
chain V and J region genes are different from Light Chain V and J
region genes and are on different chromosomes from the Light Chain
genes. (Chromosome 14 in Humans for Heavy Chain genes.
Chromosomes 2 and 22 for Light Chain genes (Kappa and Lambda)
5658 Possibilities even with no additional variations
Coupling a VH option with DH option and a JH option to a Cu gene
sequence would give a Mu Heavy chain isotype with a specific
antibody recognizing specificity.
Coupling that exact same VH-DH-JH genetic information to Cd or
Cg or Ce or Ca Heavy chain isotype gene would give the exact
same antigen specificity on a different isotype.
We would have Isotype Switching
Chain Structures of the five immunoglobulin classes in humans
(adapted from Kuby, 2nd edition)
Class
Heavy
Light
Sub-Classes
Subunit Formula
Chain
Chain
IgA
γ
α
κ or λ
γ1, γ2, γ3, γ4
γ2κ2 γ2λ2
κ or λ
α1, α2
(α2κ2)n
(α2λ2)n
n =1,2,3,4
IgM
μ
κ or λ
None
IgD
δ
κ or λ
None
δ2κ2 δ2λ2
IgE
ε
κ or λ
None
ε2κ2 ε2λ2
IgG
(μ2κ2)n
(μ2λ2)n
n = 1 or 5
Complete Heavy Chain
The puzzle of immunoglobulin gene structure
Kappa or Lambda
Light Chains
• B cells use groups of PARTS of genes to create different possible
antibodies using recombination
– Like shuffling a deck of cards, dealing out different hands
– Tightly regulated machinery controls the recombination processes
The puzzle of immunoglobulin gene structure
• B cells use groups of PARTS of genes to create
different possible antibodies using recombination
– There are variable (V), diversity (D), joining (J), and
constant (C) region gene segments
• D segments are used in antibody heavy chains only
Multigene organization of Ig genes
• Recall that Ig proteins consist of:
– two identical heavy chains
– two identical light chains
• Light chains can be either kappa or lambda
– Each set of gene families are encoded on separate
chromosomes
The mechanism of V(D)J recombination
• Five mechanisms generate antibody diversity in naïve B
cells
– Multiple gene segments―which gene segments are put together
– P nucleotide addition―templated nucleotide addition between
joints, resulting from assymetrical cleaving of hairpin structures
– Exonuclease trimming―sometimes occurs at junctions, losing
nucleotides and changing reading frames
– Non-templated N nucleotide addition―mediated by TdT activity,
adding in random nucleotides between joints
- Somatic Hypermutation
Combinatorial diversity―which heavy chains pair with which
light chains
The mechanism of V(D)J recombination
• Five mechanisms generate antibody diversity in
naïve B cells (See previous slide)
One pseudo gene
Additional sequence diversity estimated at several orders of magnitude (i.e. perhaps
1000-fold) is generated by junctional flexibility, nucleotide addition, and somatic
hypermutation. This allows for an estimated 100 million to a billion different
possible antibody seuquences in humans.
B-Cell Differenatiation
B-cell receptor expression
• Allelic exclusion ensures that each B cell synthesizes only one heavy
and one light chain.
Recombination is a very ordered process.
Nonproductive arrangements lead to programmed cell death
(apoptosis) during development.
Once one successful
heavy chain allele has
been assembled, the other
allele is shut off.
Cell then goes to making
a light chain.
Once one successful
kappa or lambda light
chain has been assembled,
the other kappa or lambda
allele is shut off.
Detailed genetic mechanisms for generating antibody diversity
are shown in Chapter 7 of Edition 7.
These specifics are beyond the scope of this introductory course
but are important for those interested in advanced work in
immunobiology or medical genetics.
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