ANTIBODY STRUCTURE AND DIVERSITY

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ANTIBODY STRUCTURE AND DIVERSITY NATURE OF THE
T AND B CELL RECEPTORS
Beverly E. Barton, Ph.D. Assistant
Professor Department of Surgery/Division of
Urology UMDNJ-NJMS MSB F672 185 S. Orange
Avenue Newark, New Jersey 07103 Telephone
973-972-0662 E-mail
bartonbe_at_umdnj.edu Telefacsimile 973-972-3892
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Igs are made by B cells. All Abs are Igs, yet
not all Igs are Abs.
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Ig SUPERFAMILY
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Ig
SUPERFAMILY Characterized by a primary sequence
of 70-110 residues in length Conserved
intrachain S-S bridges span 50-70 residues It
and other conserved residues form a tertiary
structure the Ab or Ig fold 2 basic homology
units defined from crystallographic analysis - V
and C regions Tertiary structure of V region has
9 antiparallel ß strands assume a characteristic
barrel-like structure with 2 ß sheets stabilized
by the S-S bridge S-S bridges stabilize the
basic structure, providing compact, globular
domains that are relatively insensitive to
proteolytic cleavage
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CRYSTALLOGRAPHIC RIBBON MODEL OF Ig DOMAIN
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Ab STRUCTURE, cont.
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CLASSES AND SUBTYPES OF HUMAN Ig
(species-dependent)
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Ab STRUCTURE, cont.
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Ab STRUCTURE, cont.
Cleavage of Ig with papain yields 3 fragments of
2 types
Fab has antigen (Ag) binding site. Fc C
crystal this portion of Ig 1st to be
crystallized. Fc binds to cell surface Fc
receptors, augmenting immune responses
(opsonization, e.g.).
Cleavage of Ig with pepsin yields 2 main types of
fragments
F(ab)2 2 antigen (Ag) binding sites. Fc
Largest fragment of Fc portion can
crystallize. (Smaller fragments of the Fc
also result.)
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V of both L and H Ig chains contain 3
hypervariable regions, or complementarity-determin
ing regions (CDRs). CDRs loops connecting ?
strands in Ig fold Residues in CDRs vary from
one Ab to the next, imparting Ag specificity to
each Ab. VL and VH domains at tips of Abs
packed so that 6 CDRs (3 on each) form surface
for Ag-specific binding. Residues from all 6
CDR's (VL CDR1, CDR2, CDR3 VH CDR1, CDR2, CDR3)
project from distal surface of Ab tip to bind Ag.
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2-D REPRESENTATION OF HOW CDRs JUXTAPOSE
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LYSOZYME BINDING TO ANTI-LYSOZYME
Davies, et al. J. Biol.Chem. 263 10541, 1988.
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MULTIMERIC IMMUNOGLOBULINS
IgA
J chain associates with ? or ? via S-S bonds at
the penultimate cys residue of the H chain. It is
made by B cells. Secretory component is made by
epithelial cells.
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Ab DIVERSITY
B cells are capable of making a huge
repertoire of Abs 1011 different ones. The
size of this repertoire gt the entire coding
capacity of the human genome! HOW
IS THIS POSSIBLE?????? Enormous diversity
generated from a small repertoire of coding DNA
1) somatic recombination (combinatorial
joining of gene segments) 300 V? gene
segments 4 J gene segments 30 V?
gene segments 15 D gene segments 1000 VH
gene segments 2) somatic mutation of V
gene segments 3) junctional diversity
(inaccuracies in V-J and V-D and D-J
recombination) Recombination between
V-J and V-D-J not always perfect additional
diversity arises by errors occurring
during recombination when V region moves next to
J or D regions, or D region moves next
to J region. Estimated that such inaccuracies
triple the diversity of V-J and V-D-J
joining. 4) N region insertion
At junction between D and J segments there can be
insertion of up to 15 random
nucleotides, catalyzed by terminal
deoxynucleotidyl transferase (TdT). TdT catalyzes
the polymerization of nucleotides into
DNA without a template.
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Ab DIVERSITY, cont.
1965 - Dreyer Bennett - at least 2 genes for
each polypeptide chain in Ab Proposed there must
be 100s or even 1000s of different genes for the
V regions but only 1 C region gene for each
isotype. They were partly correct. 1976 -
Tonegawa Hozumi - provided 1st experimental
proof of Dreyer Bennett hypothesis They
analyzed L chain genes from mouse myeloma DNA and
compared it to L chain genes in embryonic DNA
In differentiated Ab-producing cells (myeloma, B
cells) V C gene segments form a single V-C
segment. In embryonic DNA, V C 2 gene
segments. Ig genes undergo gene rearrangement
during B cell development. Ig gene
rearrangement - extensively studied, known to be
the primary mechanism for Ab diversity V-C
rearrangements - only known form of site-specific
DNA rearrangement in vertebrates.
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Ab DIVERSITYHOW MUCH CAN YOU GET?
Combinatorial diversity - generated by random
formation of many different VJL VDJH
combinations. Combinatorial diversity -
increased by ability of any VH region to pair
with any VL region. Random pairing of 270 VL
(30 x 5 150 possible V? 30 x 4 120 possible
V?) with 11,000 VH (65 VH x 27 DH x 5 JH)
3.5 x 106 possible Ab specificities Somatic
mutation, junctional diversity, N diversity
increase this figure by several orders of
magnitude.
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Example of actual junctional diversity found in a
series of Ig molecules in the mouse.
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EFFECT OF SEQUENCE ON Ab BINDING SITE Ag
SPECIFICITY
red CDHR3 blue CDHR2 green CDHR1 CDHR
complementarity-determining region of H chain
(hypervariable region) Effect of
insertions/deletions on Ag binding
from Lantto, J., et al. J. Biol. Chem. 2002 277
45108-45114.
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SOMATIC RECOMBINATION - H CHAIN GENE
REARRANGEMENT
Vn 1000 Dn 15
( human H chain genes)
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SOMATIC RECOMBINATION - HEAVY CHAINS
Occurs during B cell development in bone marrow.
1 DH and 1 JH spliced (D-J joining). VH
segment is spliced to DJH segment. VDJH is not
spliced to the CH segments the introns are
transcribed into primary mRNA. RNA splicing of
primary mRNA yields mature mRNA - transported to
cytoplasm translated. Leader sequence (L) at
the beginning of each VH segment - signal
sequence, which transports the new H chains to
the ER spliced off the mature H chain.
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SOMATIC RECOMBINATION - HEAVY CHAINS
V(D)J recombinase expressed in developing B cells
recognizes sequences flanking Ig gene segments.
DNA between gene segments is looped out and lost
from the DNA during V-JL or V-D-JH joining.
Splicing between gene segments is imprecise,
sometimes resulting in nonproductive
rearrangements (frame shift mutations yield stop
codons downstream and no complete H or L chain
produced). Cell dies. L leader sequence not
light chain here!
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SOMATIC RECOMBINATION
At least 2 recombination-activating gene
products, RAG-1 and RAG-2, enzymes to ligate
the DNA, TdT are required for somatic
recombination. Each B cell makes Ab of a single
Ag specificity, a single allotype (genetic
subtype). Both result from productive
rearrangement of only one L and one H chain gene.
Productive rearrangement results in successful
synthesis membrane expression of Ig.
Productive rearrangement of 1 allele blocks
rearrangement of other (allelic exclusion). If
a developing B cell does not successfully
rearrange 1 H 1 L chain genes, it dies.
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SOMATIC RECOMBINATION - L CHAIN GENE
REARRANGEMENT
Mature L chains coded for by 3 gene segments
each V, J, and C (variable region, joining
region, constant region). Mouse genome
contains 300 V, 5 J, and 1 C for ? chain only
30 V for ? chain.
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SOMATIC RECOMBINATION - LIGHT CHAINS
L chain gene segment recombination occurs similar
to H. One V? and 1 J? are spliced (V-J
joining), with removal of introns. VJ? segments
are transcribed with C? introns. RNA splicing
removes introns so that a complete ? chain is
translated. If ? chain gene segments are not
successfully rearranged, the same process occurs
with ? chain gene segments. NO D REGION IN L
CHAINS!
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LIGHT CHAIN FROM GERMLINE DNA TO B CELL Ig
germline
Ab-secreting B cell
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ISOTYPE SWITCHING IN B CELL DEVELOPMENT
Isotype switching increases functional diversity
of Ab (not Ag diversity). Occurs following Ag
stimulation requires Th2 cytokine production.
Human gene segments for CH - arranged C?, C?,
C?3, C?1, pseudogene C?, C?1, C?2, C?4, C?, C?2.
Each C gene segment (except C?) preceded by
intron containing switch region sequence. Switch
sequences differ from recombination signal
sequences found flanking V segments. Enzymes
catalyzing isotype switching not encoded by RAG1
or RAG2.
The temporal order of H chain isotype switching
activated by Ag corresponds to linear arrangement
of CH genes on chromosome.
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ISOTYPE SWITCHING IN B CELL DEVELOPMENT
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ISOTYPE SWITCHING IN B CELL DEVELOPMENT
Rearranged VDJH always expressed 1st with
membrane C? in developing B cells, with both
membrane C? and C? in mature B cells, with
secreted C? as the B cell responds to Ag. When
B cell receives proper signals from Ag
cytokines to switch to IgG3 production, eg,
recombination occurs between switch regions S?
and S?3. DNA between 2 switch sequences,
including coding sequences for ? and ? chains, is
excised. All isotype switching events
productive, since DNA splicing occurs within
introns, so no nonsense codons introduced into
transcribed DNA. Further switches to downstream
isotypes (IgA and IgE) occur if B cell receives
required cytokine signals.
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SUMMARY OF Ig GENE REARRANGEMENT IN B CELL
DEVELOPMENT
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B CELL RECEPTOR COMPLEX
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T CELL RECEPTOR GENES REARRANGEMENT
T cells with this type of receptor are called ??
T cells Majority of T cells. TCR by itself
CANNOT DOES NOT bind Ag! Instead, it
associates with other cell-surface molecules to
form TCR complex. It is monovalent no secreted
form.
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T CELL RECEPTOR GENES REARRANGEMENT
T cells with this type of receptor are called ??
T cells. Found in skin, gut, etc. Lacks common
surface markers (CD4, CD8) seen on ?? T cells ??
T cells often are cytotoxic.
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prothymocyte
immature T cell
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Mature T cells express EITHER CD4 or CD8. DO NOT
CONFUSE accessory proteins ? and ? of CD3 with
TCR chains found on ?? T cells!!!!