Immunoglobulins

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Immunoglobulins

• Generation of Diversity

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Introduction

• Immunologist estimate that each person has the
  ability to produce a range of individual
  antibodies capable of binding to a total of well
  over 1010 epitopes
• According to the germline theory, a unique gene
  encodes each antibody
• Unfortunately, for this theory to be true the
  number of antibody genes would need to be
  100-1000-fold greater than the entire human genome

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Theories

• An alternative theory, the somatic mutation
  theory, holds that a single germline
  immunoglobulin gene undergoes multiple mutations
  that generate immunoglobulin diversity. This
  scheme, however, requires an unimaginable
  mutation rate
• The immune system has developed a much more
  elegant solution- the chromosomal rearrangement
  of separate gene segments, which employs some
  elements of the germline and somatic mutation
  theories

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Gene Rearrangement

• Each light and heavy chain is encoded by a series
  of genes occurring in clusters along the
  chromosome
• In humans, the series of genes encoding the k
  light chain, ? light chain, and the heavy chain
  are located on chromosomes 2, 22, and 14
  respectively
• When a cell becomes committed to the B lymphocyte
  lineage, it rearranges the DNA, encoding its
  light and heavy chains by cutting and splicing
  together some of the DNA sequences, thus
  modifying the sequence of the variable region
  gene

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Tonegawas demonstration

• 1976used restriction enzymes and DNA probes to
  show that germ cell DNA contained several smaller
  DNA segments compared to DNA taken from developed
  lymphocytes (myeloma cells)

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Ig gene sequencing complicated the model
Structures of germline VL genes were similar for
Vk, and Vl, However there was an anomaly between
germline and rearranged DNA
Where do the extra 13 amino acids come from?
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Further diversity in the Ig heavy chain
Heavy chain between up to 8 additional amino
acids between JH and CH The D or DIVERSITY region
Each heavy chain requires three recombination
events JH to DH, JHDH to VH and JHDH VHto CH
Each light chain requires two recombination
events VL to JL and VLJL to CL
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of DNA?
2. How can the same specificity of antibody be on
   the cell surface and secreted?
3. How do V region find J regions and why dont they
   join to C regions?
4. How does the DNA break and rejoin?

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Diversity Multiple germline genes
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Diversity Multiple Germline Genes
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Reading D segment in 3 frames
Analysis of D regions from different
antibodies One D region can be used in any of
three frames Different protein sequences lead to
antibody diversity
GGGACAGGGGGC GlyThrGlyGly GGGACAGGGGGC
GlyGlnGly GGGACAGGGGGC AspArgGly
Frame 1
Frame 2
Frame 3
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Estimates of combinatorial diversity
Using functional V, D and J genes
40 VH x 27 DH x 6JH 5,520 combinations D can be
read in 3 frames 5,520 x 3 16,560
combinations 29 Vk x 5 Jk 145 combinations 30
Vl x 4 Jl 120 combinations 265 different
light chains If H and L chains pair randomly as
H2L2 i.e. 16,560 x 265 4,388,400 possibilities
Due only to COMBINATORIAL diversity
In practice, some H L combinations are
unstable. Certain V and J genes are also used
more frequently than others. Other mechanisms
add diversity at the junctions between
genesJUNCTIONAL diversity
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of
   DNA?Mathematically, Combinatorial Diversity can
   account for some diversity how do the elements
   rearrange?

1. How can the same specificity of antibody be on
   the cell surface and secreted?
2. How do V region find J regions and why dont they
   join to C regions?
3. How does the DNA break and rejoin?

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Genomic organisation of Ig genes (Numbers include
pseudogenes etc.)
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Ig light chain gene rearrangement by somatic
recombination
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Ig light chain rearrangement Rescue pathway
There is only a 13 chance of the join between
the V and J region being in frame
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Ig heavy chain gene rearrangement
Somatic recombination occurs at the level of DNA
which can now be transcribed
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RNA processing
The Heavy chain mRNA is completed by splicing the
VDJ region to the C region
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of
   DNA?Combinatorial Diversity and genomic
   organisation can account for some diversity

1. How can the same specificity of antibody be on
   the cell surface and secreted?
2. How do V region find J regions and why dont they
   join to C regions?
3. How does the DNA break and rejoin?

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Remember These Facts?

• Cell surface antigen receptor on B cells
• Allows B cells to sense their antigenic
  environment
• Connects extracellular space with intracellular
  signalling machinery
• Secreted antibody functions
• Neutralisation
• Arming/recruiting effector cells
• Complement fixation

How does the model of recombination allow for two
different forms of the same protein?
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The constant region has additional, optional
exons
h
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Membrane IgM constant region
mRNA
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Secreted IgM constant region
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Alternative RNA processing generates
transmembrane or secreted Ig
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(a)
Secreted membrane forms of the heavy chain by
alternative ( differential ) RNA processing of
primary transcript.
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Synthesis, assembly, and secretion of the
immunoglobulin molecule.
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of
   DNA?Combinatorial Diversity and genomic
   organisation accounts for some diversity

1. How can the same specificity of antibody be on
   the cell surface and secreted?Use of alternate
   polyadenylation sites

1. How do V region find J regions and why dont they
   join to C regions?
2. How does the DNA break and rejoin?

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V, D, J flanking sequences
Sequencing up and down stream of V, D and J
elementsConserved sequences of 7, 23, 9 and 12
nucleotides in an arrangement that depended upon
the locus
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Gene rearrangements are made at recombination
signal sequences (RSS). RSSs are heptamer-nonamer
sequences
Each RSS contains a conserved heptamer, a
conserved nonamer and a spacer of either 12 or 23
base pairs.
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There is a RSS downstream of every V gene
segment, upstream of every J gene segment and
flanking every D gene segment
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Recombination signal sequences (RSS)
12-23 RULE A gene segment flanked by a 23mer
RSS can only be linked to a segment flanked by a
12mer RSS
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1. Rearrangements only occur between segments on
the same chromosome. 2. A heptamer must pair
with a complementary heptamer a nonamer must
pair with a complementary nonamer. 3. One of the
RSSs must have a spacer with 12 base pairs and
the other must be 23 base pairs (the 12/23 rule).
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Molecular explanation of the 12-23 rule
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Molecular explanation of the 12-23 rule
Loop of intervening DNA is excised

• An appropriate shape can not be formed if two
  23-mer flanked elements attempted to join (i.e.
  the 12-23 rule)

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Junctional diversity
Mini-circle of DNA is permanently lost from the
genome
Imprecise and random events that occur when the
DNA breaks and rejoins allows new nucleotides to
be inserted or lost from the sequence at and
around the coding joint.
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Non-deletional recombination
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Non-deletional recombination
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of
   DNA?Combinatorial Diversity and genomic
   organisation accounts for some diversity

1. How can the same specificity of antibody be on
   the cell surface and secreted?Use of alternative
   polyadenylation sites

1. How do V region find J regions and why dont they
   join to C regions?The 12-23 rule

1. How does the DNA break and rejoin?

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Steps of Ig gene recombination
Recombination activating gene products, (RAG1
RAG 2) and high mobility group proteins bind to
the RSS
The two RAG1/RAG 2 complexes bind to each other
and bring the V region adjacent to the DJ region

• The recombinase complex makes single stranded
  nicks in the DNA. The free OH on the 3 end
  hydrolyses the phosphodiester bond on the other
  strand.
• This seals the nicks to form a hairpin structure
  at the end of the V and D regions and a flush
  double strand break at the ends of the heptamers.
  
• The recombinase complex remains associated with
  the break

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Steps of Ig gene recombination
A number of other proteins, (Ku70Ku80, XRCC4 and
DNA dependent protein kinases) bind to the
hairpins and the heptamer ends.
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Junctional diversity P nucleotide additions
The recombinase complex makes single stranded
nicks at random sites close to the ends of the V
and D region DNA.
The 2nd strand is cleaved and hairpins form
between the complimentary bases at ends of the V
and D region.
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Heptamers are ligated by DNA ligase IV
V and D regions juxtaposed
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Generation of the palindromic sequence
Endonuclease cleaves single strand at random
sites in V and D segment
The nicked strand flips out
In terms of G to C and T to A pairing, the new
nucleotides are palindromic. The nucleotides GA
and TA were not in the genomic sequence and
introduce diversity of sequence at the V to D
join.
(Palindrome - A Santa at NASA)
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Junctional Diversity  N nucleotide additions
Terminal deoxynucleotidyl transferase (TdT) adds
nucleotides randomly to the P nucleotide ends of
the single-stranded V and D segment DNA
CACTCCTTA
TTCTTGCAA
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Generation of Antibody Diversity
P-nucleotide and N-nucleotide addition during
joining.
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P and N region nucleotide alteration adds to
diversity of V region

• During recombination some nucleotide bases are
  cut from or add to the coding regions (p
  nucleotides)
• Up to 15 or so randomly inserted nucleotide bases
  are added at the cut sites of the V, D and J
  regions (n nucleotides_
• TdT (terminal deoxynucleotidyl transferase) a
  unique enzyme found only in lymphocytes
• Since these bases are random, the amino acid
  sequence generated by these bases will also be
  random

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Junctional Diversity
TTTTT TTTTT TTTTT
Germline-encoded nucleotides
Palindromic (P) nucleotides - not in the germline
Non-template (N) encoded nucleotides - not in the
germline
Creates an essentially random sequence between
the V region, D region and J region in heavy
chains and the V region and J region in light
chains.
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Problems?

1. How is an infinite diversity of specificity
   generated from finite amounts of
   DNA?Combinatorial Diversity, genomic
   organisation and Junctional Diversity

1. How can the same specificity of antibody be on
   the cell surface and secreted?Use of alternative
   polyadenylation sites

1. How do V region find J regions and why dont they
   join to C regions?The 12-23 rule

1. How does the DNA break and rejoin?Imprecisely to
   allow Junctional Diversity

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Why do V regions not join to J or C regions?
IF the elements of Ig did not assemble in the
correct order, diversity of specificity would be
severely compromised
Full potential of the H chain for diversity needs
V-D-J-C joining - in the correct order
Were V-J joins allowed in the heavy chain,
diversity would be reduced due to loss of the
imprecise join between the V and D regions
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Additional Degrees of Variation

• Somatic hypermutation Stimulated memory B cells
  accumulate small mutations on the VL or VH
  leading to affinity maturation to antigens that
  are frequently or chronically present
• Isotype switching

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Somatic hypermutation
What about mutation throughout an immune response
to a single epitope? How does this affect the
specificity and affinity of the antibody?
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Somatic hypermutation leads to affinity maturation
Cells with accumulated mutations in the CDR are
selected for high antigen binding capacity thus
the affinity matures throughout the course of the
response
Lower affinity - Not clonally selected
Higher affinity - Clonally selected
Identical affinity - No influence on clonal
selection
Hypermutation is T cell dependent Mutations
focussed on hot spots (i.e. the CDRs) due to
double stranded breaks repaired by an error prone
DNA repair enzyme.
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Allelic Exclusion

• A single B cell can express only one VL and one
  VH allele to the exclusion of all others
• Both must be on the same member of the chromosome
  pair-either maternal or paternal
• The restriction of VL and VH expression to a
  single member of the chromosome pair is termed
  allelic exclusion
• The presence of both maternal and paternal
  allotypes in the serum reflects the expression of
  different alleles by different population of B
  cells

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Allelic exclusion only one chromosome is active
in any one lymphocyte
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Antibody isotype switching
Throughout an immune response the specificity of
an antibody will remain the same (notwithstanding
affinity maturation) The effector function of
antibodies throughout a response needs to change
drastically as the response progresses. Antibodie
s are able to retain variable regions whilst
exchanging constant regions that contain the
structures that interact with cells.
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Switch regions

• Upstream of C regions are repetitive regions of
  DNA called switch regions. (The exception is the
  Cd region that has no switch region).

• The Sm consists of 150 repeats of
  (GAGCT)n(GGGGGT) where n is between 3 and 7.
• Switching is mechanistically similar in may ways
  to V(D)J recombination.
• Isotype switching does not take place in the bone
  marrow, however, and it will only occur after B
  cell activation by antigen and interactions with
  T cells.

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7 means of generating antibody diversity
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Generation of Antibody Diversity

• Germ line diversity.
• Combinatorial diversity.
• Junctional diversity.
• Somatic hypermutation ( affinity maturation)