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Chapter 2
Antibody Structure and the
Generation of B-Cell Diversity
The Structural Basis of
Antibody Diversity
Antibodies and Antigens
• Antibodies….
 Five chemically and physically distinct classes of
antibodies (IgG, IgA, IgM, IgD, IgE).
 Bind with high specificity and affinity.
 Produced by the B lymphocytes in response to
infection.
 Circulate as a major component of the plasma in
blood and lymph.
 Bind to pathogenic microorganism and their toxins
in the extracellular spaces of the body.
 Antibody repertoire might be as high as 1016.
• Antigens….
 Are bound by antibodies.
 Are biological macromolecules.
• proteins and carbohydrates most common antigens
Antibody (Ab) - Immunglobulins
• Antibodies are the secreted form of proteins known more
generally as immunoglobulins (Igs)
• Mature B cells express Igs (B cell receptor)
• When Ag binds Igs, the B cell proliferates and then
differentiates into a plasma cell
• Plasma cell secretes Ab with same specificity as the
membrane-bound Igs
Antibody Functions
Antibody functions in host defense:
1. Antibody recognizes and binds its
corresponding antigen
2. Antibody targets the bound antigen to
other components of the immune system
• each of these functions is performed
by different parts of the antibody
molecule
Antibody Structure
• Antibodies are glycoproteins
• Basic unit of four polypeptide
chains (2 pairs L & H chains)
– covalent disulfide bridges
– noncovalent interactions
• Total MW~150kDa
– Two identical heavy chains (H
chains) (50 kDa each)
– Two identical, smaller, light
chains (L chains) (25 kDa each)
• Arms of Y
– L chains paired with N-terminal
pair of H chain - Covalently
linked by a disulfide bond.
• Stem of the Y
– consists of the paired C-terminal
portions of the two-H chains
Antibody Variability
• H and L chains are divided into V
and C regions
• The V region
– variability = great diversity
– concentrated in the N-terminal
region
– paired V regions of a H and L
chain
• form the antigen binding
site
• two identical antigen
binding sites
• The C region
– constant region
– determines the fate of the
antigen
– less variation in amino acid
sequence between antibodies
N-terminal
C-terminal
Antibody Affinity
Affinity is the tightness of binding of an
antibody binding site to an antigen.
The tighter the binding, the less likely the
antibody is to dissociate from antigen.
Different antibodies to the same antigen
vary considerably in their affinity for an
epitope.
Antibodies produced by a memory
response have higher affinity than those
in a primary response.
Antibody Valence and Avidity
• Valence of an antibody is the maximum number of
antigenic determinants with which an antibody can react.
 Multiple binding sites on an antibody dramatically
increases its binding (avidity) to antigens on particles
such as bacteria or virus.
• Example: two binding sites on IgG are 100 times more
effective at neutralizing virus than two unlinked binding
sites.
 Avidity is the firmness of association between a
multideterminant antigen and the antibodies
produced against it.
 This combined effect (avidity) results from synergy of
the binding strengths of each binding site.
Antibody Valence and Avidity
Proteases Cleave Antibody into Fab and
Fc Fragments
• Papain cleaves the Ig
molecule into three pieces:
 Two Fab (Fragment Antigen
Binding) fragments
 Antigen binding part
 One Fc fragment (Fragment
Crystallizable)
 Part responsible for effector
functions.
• Pepsin cleaves IgG to yield 1
F(ab’)2 fragment and the Fc
fragment
 Fc fragment is broken into a
number of smaller pieces
Antibody Classes
• Different antibody classes with different biological
activities have evolved to deal with diverse antigens.
• Heavy-chain constant regions define five main
isotypes.
Heavy chains , , , , 
IgA, IgD, IgE, IgG and IgM
Each isotype has a different effector functions –
with some overlap.
Example: IgA is the most common antibody in
mucosal secretions while IgM is mainly found in
the plasma, and both are most effective at those
locations
Isotypes of Heavy and Light
Chains
• Heavy-chain constant regions define five main isotypes
of antibodies: IgA, IgD, IgE, IgG, and IgM
– Differences in length and sugars of heavy chain
• Only two isotypes of light chain
– kappa () and lambda ()
– Each antibody has either  or  chains
• The antigen binding site is composed of two variable
domains (VH and VL)
– Constant domain of light chain is CL
– Constant domain of the heavy chain is CH
• The  heavy chain of IgG has three domains - CH1, CH2,
CH3
• Some other isotypes have four C domains
IgG (MW~150 kD)
• Four different subclasses (IgG1, IgG2, IgG3 and IgG4)
– Each subclass has slightly different H-chains and
corresponding differences in their function.
• Found both in vascular and extravascular spaces as well as
in secretions.
• Most abundant Ig in the blood.
• Provide the bulk of immunity to most blood borne infectious
agents.
• The only antibody class to cross the placenta to provide
passive humoral (innate) immunity to the developing and
newborn infant.
IgA (In Serum~170 kD, External
Secretions ~420 kD Dimer)
• Major Ig present in external secretions - colostrum, milk and saliva.
• In addition to the L-chains and the IgA heavy chain () secreted IgA also
contains two other polypeptide chains– secretory component (SC) and J-chain (joining chain)
• SC is part of the molecule (poly-Ig receptor) involved in the transepithelial transport
of exocrine IgA and stabilizes IgA against proteolytic degradation.
• The two four-chain units composing secretory IgA are held together by the J-chain
through disulfide bridges.
• A first line of defense against microbes entering through mucosal surfaces.
– Respiratory, gastrointestinal and genitourinary tracts
• Secretory (dimeric) IgA is synthesized locally by plasma cells in mammary
and salivary glands and along the respiratory, gastrointestinal and
genitourinary tracts.
• IgA prevents colonization of mucosal surfaces by pathogens and mediates
their phagocytosis.
(1)
Protected from protease
(5)
(2)
(3)
Receptor-mediated
endocytosis
(4)
IgM (~900 kD)
• IgM is the first antibody produced by B cells.
• Is the antigen receptor (B cell receptor).
– Expressed as a four-chain unit - two  H-chains and
two L-chains.
• Also present as a soluble molecule in the blood.
– J-chain is associated with IgM in the blood and initiates
the polymerization of its subunits.
• In the circulation, IgM is composed of five four-chain units
with 10 combining sites (recall avidity slide).
– Has high avidity for antigens and is very efficient per
molecule in dealing with pathogens especially early in
the immune response before sufficient quantities of IgG
have been produced.
IgD
• Present in low quantities in the circulation
– (0.3 mg/ml in adult serum)
• IgD functions primarily as an antigen receptor on B cells.
– B cells express both IgM and IgD that are specific for
the same antigen.
– When IgM and IgD on a B cell interact with an antigen,
the antigen is internalized and processed and
presented to helper T cells.
– This triggers B cells to proliferate and differentiate into
plasma cells, thus initiating the development of a
humoral immune response
– Humoral-pertains to extracellular fluid including the
plasma and lymph.
IgE
• Present in serum at very low levels
– nanograms per milliliter
– Plays significant roles in
• enhancing acute inflammation
• protection from infection by worms
• Allergic reactions are predominantly associated with IgE
– After antigen stimulation induces plasma cells to
produce IgE
• IgE binds to receptors on mast cells which are
specific for the Fc regions of IgE.
• Antigen reintroduced into a previously sensitized
individual binds to IgE on ‘armed’ mast cells and
triggers release of the pharmacologically active
agents – histamine (involved in immediate
hypersensitivity such as hay fever and asthma).
Structural Organization of Human
Immunoglobulin Isotypes
• Differences in length of the
heavy-chain C regions and
locations of the disulfide bonds.
• The hinge region present in IgG,
IgA, IgD but not in IgM, IgE.
• Isotypes differ in the distribution
of N-linked carbohydrate groups
• All occur as monomers in their
membrane-bound form.
• Soluble, secreted form of IgD,
IgE and IgG are always
monomers.
• IgA forms monomers and
dimers.
• IgM forms pentamers.
Immunoglobulins are flexible
• The hinge region is a
flexible tether, allowing
independent
movement of the two
Fab arms.
• The angle between the
arms can vary from 0°
in dimers, to 60° or 90°
degrees in triangles or
squares.
Immunoglobulin Chains are Folded
into Compact and Stable protein
Domains
• The V region at the N-terminal end of each H or L chain is composed of
a single variable domain (V domain, VH or VL).
– VH and VL domains together form an antigen-binding site.
• The other domains have little or no sequence diversity within a particular
isotype and are termed the constant domains (C domains) .
– Constant region of a light chain is composed of a single CL domain
– Constant region of a H chain is composed of three or four C domains
(isotype dependent)
• the  heavy chains of IgG have 3 domains-Ch1, CH2, Ch3
• other isotypes have four C domains (previous slide)
– In the complete IgG molecule the pairing of the four polypeptide
chains produces three globular regions, corresponding to the two
Fab arms and the Fc stem (refer to fig 2.5)
Immunoglobulin Chains are
Folded into Compact and Stable
protein Domains
Immunoglobulin-like domain = Immunoglobulin superfamily
Ag-binding Site is Formed from the
Hypervariable Regions of a Heavy
Chain and a Light Chain V Domain
• V domains of light and
heavy chains have
hypervariable regions
(HV), flanked by
framework regions.
• Three hypervariable
regions in each domain.
The Hypervariable Regions of Antibody
V Domains Lie in Discrete Loops at
One End of the Domain Structure
• The hypervariable regions of antibody
V domains lie in discrete loops at the
end of the domain.
• The pairing of the heavy and light
chains bring together the
hypervariable loops and create a
composite hypervariable surface that
forms the antigen-binding site.
• This surface determines specificity
and diversity.
• The hypervariable loops are also
called the complementaritydetermining regions (CDRs).
Epitopes for Antibody Binding are
Exposed
Poliovirus
• Antibodies that are most
effective are those that bind
the exposed surface of a
pathogen.
• Epitope is the part of the
antigen to which an antibody
binds.
• An antigen that contains
more than one epitope is
called multivalent.
Physical Properties
of Antibody Binding Sites
• The antibody binds to an antigenic determinant or epitope,
commonly: glycoproteins, polysaccharides, glycolipids, and
proteoglycans.
• Epitopes can be linear, or
discontinuous (when
different parts of the antigen
are folded together).
• The binding forces are
non-covalent: electrostatic,
hydrogen bonds, van der
Waals forces and
hydrophobic forces.
• The binding strength is known as the affinity of the antibody
for the antigen.
Epitopes Can Bind to Pockets,
Grooves or Extended Surfaces in
the Antibody Binding Sites
• The type of antigen bound by an antibody
depends on the shape of the antigen-binding site.
 Antigens that are small molecules can be bound within
deep pocket.
 Linear epitopes from proteins or carbohydrates can be
bound within clefts or grooves.
 Binding of conformation epitopes of folded proteins
takes place over an extended surface area.
Antibodies Binding to Three different
Types of Epitope
Peptide epitope (red)
bound to pocket in
CDR loops
Peptide epitope in
groove between two
V domains
Fab binding to
surface epitope of
lysozyme
Monoclonal Antibodies
• 1975 Kohler and Milstein developed a procedure to create
cell lines producing predetermined, monospecific and
monoclonal antibodies (mAb).
• Standardized procedures involving fusion of an immortal
cell (i.e. myeloma tumor cell) with a specific
predetermined antibody-producing B cell from immunized
animals or humans to create hybridoma cells producing
monospecific and monoclonal antibodies (mAb).
• Monoclonal antibodies important in a variety of
therapeutic, diagnostic and research settings.
Production of Monoclonal Antibodies
• Produced from a
single clone from
antibody-producing
cells and thus have
identical Ag-binding
sites and identical
isotypes.
• Antibodies can be
humanized (mouse
CDRs into human
antibody) for
therapeutic purposes.
The flow
cytometer
allows
individual cells
to be identified
by surface
molecules.
Generation of
Immunoglobulin Diversity
• In all cells (except B cells) Ig genes are in a fragmented
form that cannot be expressed.
• The Ig H-chain and L-chain loci consists of families of gene
segments containing alternative versions of parts of the Ig
variable region.
• Germ-line DNA contains multiple gene segments encoding
portions of a single Ig heavy or light chain.
Germ-line DNA
Rearranged Functional DNA
• In B cell germ-line DNA gene segments are rearranged
into functional genes.
• Mature B cells contain chromosomal DNA that is no longer
identical to germ-line DNA.
• Genomic rearrangement is an essential feature of
lymphocyte differentiation.
Germ-line DNA
Rearrangement
Rearranged Functional DNA
Generation of Diversity:
Antibody Genes
• DNA encoding Igs is found in three unlinked gene groups.
– One group encodes kappa () L-chains
– One lambda () L-chains
– One H-chains
• Each L-chain gene groups has multiple different copies of V
gene segments and J segments.
• In addition, in the kappa () chain group there is one gene
segment encoding the constant regions of kappa () chains.
• In the lambda () groups there are four lambda () chain
constant regions gene segments.
• The H-chain gene groups has multiple different copies of V,
D, and J gene segments and one gene segment for each of
the constant regions for the different antibody classes and
subclasses.
Generation of Diversity:
Gene Rearrangement
• During development, a single B cell randomly selects
from its H-chain gene group, one V, one D and one J
gene segment for rearrangement.
• The B cell then selects a L-chain from the kappa ()
or lambda () gene group, selecting one V and one
gene J gene segment for rearrangement.
• These gene segments then recombine to create a
gene (VJ) encoding the L chain variable region and a
gene (VDJ) encoding the H chain variable region.
Generation of Ig Diversity in B Cells
Prior to Contact with Antigen
• In the germ line configuration, the Ig genes are
fragmented into gene segments along the chromosome.
• Individual gene segments must be rearranged.
• This only occurs in B cells.
• It occurs during their development from precursors in the
bone marrow.
• When gene rearrangements complete, heavy and light
chains are produced and membrane bound Ig is
expressed on the cell surface.
• The B cell can now recognize and respond to Ag.
Chromosomal Arrangement
of Ig Genes
• Heavy-chain locus on chromosome 14;  lightchain on chromosome 2;  light-chain on
chromosome 22.
• Different segments encode leader peptide (L),
variable region (V) and constant regions (C).
• Heavy-chain locus contains C genes for all
isotypes.
• The C genes are ready to be transcribed.
• The V genes first undergo rearrangement.
Immunoglobulin Heavyand Light–Chain Loci
• The V regions of light-chain comprised of two segments:
variable (V) and joining (J).
• Heavy chain also contains D segments between V and J.
• First and second hypervariable region of light-chain (CDR1, 2)
are encoded by different V segments, the third CDR is
determined by the V/J junction.
The Germline Organization of the Human
Immunoglobulin Heavy-Chain and LightChain Loci
Somatic Recombination of Ig
All cells
B cells
• V-region sequences
are constructed from
gene segments
• L-chain, single
recombination VJ
• H-chain, 2
recombinations: first
DJ, then VDJ.
• C-regions at not
rearranged at this
point.
#’s of Functional Gene Segments
Used to Construct the Variable
Regions of Human Ig H and L Chains
• The V, J (and D) segments are
combined at random, thus
many variations possible.
• Human  light chain: 40 V
segments and 5 J segments therefore 200 combinations
(40 x 5).
• Human  light-chain: 30 x 4 =
120.
• Human heavy-chain: 65 V, 27
D, 6 J (65 x 27 x 6 = 10,530
combinations).
Mechanism of V-region DNA
Rearrangements
• There are unique recombination signal
sequences (RSSs) flanking each germ-line V,
D, and J gene segment.
• One RSS is located 3’ to each V gene
segment and 5’ to each J gene segment, and
on both sides of each D gene segment.
• These sequence are signals for the
recombination process that rearranges the
gene.
Each RSS contains conserved palinodromic heptamer
and a conserved AT-rich nonamer sequence, separated
by an intervening sequence of 12 or 23 base pairs.
The intervening base pairs correspond to one or two
turns of the DNA helix.
Two turns of DNA helix
One turn of DNA helix
Each V, D, Or J Gene Segment is Flanked
by Recombination Signal Sequences
2-turn RSS
1-turn RSS
2-turn RSS
1-turn RSS
2-turn RSS
1-turn RSS
Signal sequences
having 1-turn spacer
can only join with
sequences having
2-turn spacers –
1-turn/2-turn joining
rule.
1-turn RSS
2-turn RSS
Recombination Enzymes
• Set of enzymes needed to recombine V, D, and
J gene segments is called V(D)J recombinase.
• Two components of the recombinase – RAG-1
and RAG-2 are only made in lymphocytes.
• Other enzymes required are present in all cells:
DNA ligase IV, DNA-dependent protein kinase
(DNA-PK) and Ku protein.
• First step in gene rearrangement is the binding
of the RAG complex to the RSS.
Gene Segments Encoding the Variable
Region are Joined by Recombination at
Recombination Signal Sequences
• A RAG complex binds to the 23-bp spacer (2-turn RSS)
and another to the 12-bp spacer (1-turn-RSS), so that a
RSS containing a 12-bp spacer is brought together with
that containing a 23-bp spacer.
1-turn/2-turn joining Rule
(12/23 Rule)
• 12/23 rule ensures
that gene segments
are joined in the
correct order.
• The DNA molecules
are broken at the
ends of the
heptamer
sequences (orange)
and are then joined
together.
Different Topologies,
Signal and Coding Joints
• The DNA molecules are broken
at the ends of the heptamer
sequences (orange) and are
then joined together with differ
topologies.
• The region of DNA that was
originally between the V and J
segments to be joined is
excised as a small circle of
DNA that has no function
(Signal joint).
• Within the chromosomal DNA,
the V and J segments are
joined to form the coding joint.
Recombination Enzymes Produce
Additional Diversity in Ag-binding Sites
• The enzymes that cut
and splice DNA
during recombination
introduce additional
nucleotides not
encoded in germ line
DNA.
• Known as junctional
diversity.
• Important source of
Ig variability - adds a
factor of 3 x 107 to
overall diversity.
If one allele
rearranges nonproductively, then
the B cell
rearranges the other
allele.
If this also fails and
no rearranged
heavy or light chain
results the cell will
die.
B cells are diploid and contain both maternal and paternal
chromosomes. Rearranged heavy chain or light is expressed
from only one chromosome. This is called allelic exclusion.
Diversity of Immunoglobulin is
Generated by Multiple Processes
• Multiple germ-line gene segments
• Combinatorial V-(D)-J joining
Subsequent joining of coding sequences is
imprecise.
• Junctional diversity
• P-region nucleotide addition
• N-region nucleotide addition
• Somatic hypermutation
• Combinatorial association of heavy and light
chains
Mature B cells produces
both IgM and IgD with
identical antigen
specificity.
Naïve B Cells Use Alternative
Splicing to Make Both IgM and IgD
• Rearrangement of V, D, and J segments produces a
functional heavy chain gene.
• The same assembled V-region serves all the heavy-chain
C genes.
• The B cell first expresses IgM and IgD ( and  nearest to
assembled VDJ).
Expression of IgM and IgD
• Transcription begins with VH promoter and extends
through  and  C genes.
• This long primary transcript is processed by cleavage,
polyadenylation and splicing.
• Cleavage and polyadenylation of the  site (pA) and
splicing between C exons yields mRNA encoding 
heavy chain for IgM.
• Cleavage and polyadenylation of the  site (pA) and
splicing between C exons yields mRNA encoding 
heavy chain for IgD.
• Upon encountering a specific antigen, many B cells
change the isotype that they produce and produce IgG,
or IgA, or IgE.
Expression of IgM and IgD
The Functional B Cell
Receptor
• Membrane-bound Igs are
associated with two other
proteins: Ig and Ig
• Ig and Ig have longer
cytoplasmic tails than Ig and
interact with intracellular
signaling molecules
Diversification of Antibodies
After
B Cells Encounter Ag
A B cell’s first encounter with antigen triggers
proliferation, differentiation and secretion of
antibodies.
Production of Secreted
Antibodies
• Gene rearrangement in immature B cell leads to expression
of functional heavy and light chains.
• Mature (naïve) B cell expresses surface IgM and IgD.
• After encountering Ag, IgM, and IgD produced as secreted
Abs. IgM important; IgD has unknown function.
• All classes of Ig can be made as membrane form and
secreted form (the membrane binding domain edited out by
alternative splicing).
• Mature plasma cell only produces secreted antibody.
Production of Secreted Antibodies
• Difference
between
membrane-bound
and secreted
antibody lies in the
Carboxy-terminus
of H-chain.
• Secreted antibody
has a hydrophilic
sequence.
Production of Membrane Antibodies
• Membraneassociated
antibody has a
hydrophobic
anchor sequence
that is inserted into
membranes.
• Difference is
determined by
different pattern of
RNA processing.
Expression of the membrane or secreted
forms of  and  heavy chains depends on
polyadenylation sites.
Secreted IgM
Membrane IgM
Expression of the membrane or secreted
forms of  and  heavy chains depends on
polyadenylation sites.
Secreted IgM
Membrane IgD
Rearranged V-region Sequences
Further Diversified by Somatic
Hypermutation
• Once B cell has been activated by Ag, further
diversification of the V-domain coding region occurs.
• Called somatic hypermutation - introduces point
mutations throughout rearranged V regions at very high
rate – more than a million times greater than the ordinary
mutation rate.
• Acts selectively at certain types of DNA sequences
commonly found in CDRs.
• Mechanism involves activation-induced cytidine
deaminase (AID) – an ezyme that converts cytidine to
uracil, which are the excised and replaced with nontemplated nucleotides.
Somatic Hypermutation
Introduces Diversity into Expressed
Immunoglobulin Genes
• Antibodies, raised by immunization with the same antigen,
were collected 1 and 2 weeks after immunization and
amino acid sequences were determined.
• Each red line represents one antibody and the red bars
represent amino acid positions that differ from the
prototypic sequence.
• One week after primary immunization most of the
antibodies were IgM and showed very little sequence
variation in the V region.
• Two weeks after immunization, both IgG and IgM were
represent and all six CDRs were affected.
1 Week Versus 2 Weeks Post
Primary Immunization
Affinity Maturation
• Thus somatic hypermutation results in mutant Igs on the
surface of B cells.
• Some of these mutants will have a higher affinity for the
Ag - they will therefore be more likely to bind the Ag, and
B cells bearing these molecules will be preferentially
selected to mature into Ab-secreting cells.
• As immune response proceeds, antibodies of higher
affinity will be produced.
• Called “affinity maturation”
Isotype Switching Produces Igs with
Different C Regions but Identical Ag
Specificities
• IgM is the first Ab produced in an immune response.
• Secreted as a circular pentamer; has 10 binding sites; therefore
binds tightly to Ag; limited in effector mechanisms.
Switch Sequences or Switch Regions
• Antibodies with other effector mechanisms are produced
by isotype switching or class switching.
• Involves a further DNA recombination event that places
the original V-region with other heaving C-regions.
• Switching is accomplished by recombination within a
cluster of C genes, excising the previous C genes and
joining a new C gene with previously assembled Vregion.
• Antigen specificity thus remains the same but the
isotype of the antibody changes.
• Flanking the 5’ side of each C gene, with the exception
of the  gene, are highly repetitive sequences that
mediate recombination – called switch regions or
sequences.
Switch Sequences or Switch Regions
The Physical Properties of the
Human Immunoglobulin Isotypes
• Antibodies with different constant regions have different effector
functions.
• Humans have five classes of Ig: IgA, IgD, IgE, IgG, and IgM.
• Classes further divided into subclasses: IgG1, IgG2, IgG3, IgG4.
Antibodies Act on Pathogens
in Various Ways
• Neutralizing antibodies directly inactivates
pathogen.
– For example, virus prevented from binding its receptor
• Opsonization - facilitate phagocytosis.
• IgM first antibody produced against pathogen low affinity, therefore multiple binding sites
needed for strong binding.
• Ag binding exposes sites on the C region that
permit complement fixation.
• This results in opsonization or direct killing by
complement.
IgM
• First antibody
produced.
• Principly made by
plasma cells in
lymphoid organs.
• On initial immune
response most of the
antibody will be of
low affinity and the
multi-binding sites on
IgM are needed.
Somatic Hypermutation Leads to
Antibodies with Increased Affinity
• After affinity maturation, two Ag binding sites
are sufficient to produce strong binding.
• By switching isotype, different effector functions
are brought into play while preserving antigen
specification.
IgG
• IgM gives way to IgG
• IgG most abundant
antibody in the internal
fluids.
• IgG more flexible.
• Also implement more
effector functions.
• IgG bound by Fc receptors,
can activate complement,
can cross the placenta to
provide fetus protective
antibodies from mother.
There are four human IgG subclasses,
distinguished by the  chain sequences.
IgA
• Monomeric IgA found in the
circulation.
• Dimeric IgA is made in lymphoid
tissue underlying mucosal surfaces
and is the antibody secreted into the
lumen of the gut.
• The principle antibody in secretions,
including milk, tears, saliva and
sweat. Mucosa is a major area of
intersection between pathogens and
the immune system.
• More IgA is made than any other
isotype.
IgE
• IgE binds to high affinity
receptor on mast cells.
• Ag binding provokes a
strong inflammatory
reaction.
• Involved in expulsion of
worms and other
parasites.
• Triggers allergic reactions.
Each Immunoglobulin Isotype has
Specialized Functions and Distinct
Properties
Dealing with Chapter 2
OVERVIEW
Proteases Cleave Antibody into Fab and
Fc Fragments
• Papain cleaves the Ig
molecule into three pieces:
 Two Fab (Fragment Antigen
Binding) fragments
 Antigen binding part
 One Fc fragment (Fragment
Crystallizable)
 Part responsible for effector
functions.
• Pepsin cleaves IgG to yield 1
F(ab’)2 fragment and the Fc
fragment
 Fc fragment is broken into a
number of smaller pieces
Structural Organization of Human
Immunoglobulin Isotypes
• Differences in length of the
heavy-chain C regions and
locations of the disulfide bonds.
• The hinge region present in IgG,
IgA, IgD but not in IgM, IgE.
• Isotypes differ in the distribution
of N-linked carbohydrate groups
• All occur as monomers in their
membrane-bound form.
• Soluble, secreted form of IgD,
IgE and IgG are always
monomers.
• IgA forms monomers and
dimers.
• IgM forms pentamers.
Antibodies Binding to Three different
Types of Epitope
Peptide epitope (red)
bound to pocket in
CDR loops
Peptide epitope in
groove between two
V domains
Fab binding to
surface epitope of
lysozyme
The Germline Organization of the
Human Immunoglobulin Heavy-Chain
and Light-Chain Loci
Somatic Recombination of Ig
• V-region sequences
are constructed from
gene segments
• L-chain, single
recombination VJ
• H-chain, 2
recombinations: first
DJ, then VDJ
Each V, D, Or J Gene Segment is Flanked
by Recombination Signal Sequences
• Two types of RSS exist
• One consist of a nonamer (9bp) and a heptamer (7bp) separated by a
spacer of 12bp
• The other consists of the same 9- and 7-bp separated by a 23-bp spacer
Recombination Enzymes Produce
Additional Diversity in Ag-binding Sites
• The enzymes that cut
and splice DNA during
recombination introduce
additional nucleotides
not encoded in germ
line DNA
• Known as junctional
diversity
• Important source of Ig
variability - adds a
factor of 3 x 107 to
overall diversity
Expression of IgM and IgD
Transcription begins with VDJ and includes  and  C genes.
This primary RNA transcript is spliced to produce IgM or IgD mRNA
Naïve B cells express both IgM and IgD on their surfaces
Upon encountering a specific antigen, many B cells change the isotype
that they produce
• They stop making IgM and IgD, and now produce IgG, or IgA, or IgE
•
•
•
•
The Functional B Cell
Receptor
• Membrane-bound Igs are
associated with two other
proteins: Ig and Ig
• Ig and Ig have longer
cytoplasmic tails than Ig and
interact with intracellular
signaling molecules
Affinity Maturation
• Thus somatic hypermutation results in mutant Igs on the
surface of B cells
• Some of these mutants will have a higher affinity for the
Ag - they will therefore be more likely to bind the Ag, and
B cells bearing these molecules will be preferentially
selected to mature into Ab-secreting cells
• As immune response proceeds, antibodies of higher
affinity will be produced
• Called “affinity maturation”
Production of Membrane or Secreted
Antibodies by Alternative RNA
Processing
Switch Sequences or Switch
Regions
• Isotype switching - a recombination within the cluster of C genes that
excises the previously expressed C gene and brings a different one into
juxtaposition with the assembled V-region sequence
• Antigen specificity of the Ab remains unchanged, the isotype changes
• Flanking the 5’ side of each C gene, with the exception of the gene are
highly repetitive sequences that mediate recombination
Each Human Immunoglobulin Isotype has
Specialized Functions and Distinct
Properties