BIOL 4345 FA22 chapter 9 lecture slides.pdf

Biol

1. © 2022 Oxford University Press
Immunoglobulins
and B-Cell
Diversity
Chapter 9

2. © 2022 Oxford University Press
Chapter 9
Immunoglobulins
and B-Cell
Diversity
9.1 What is the structure of immunoglobulins? 9.1 What is the structure of immunoglobulins? 9.2 How does genetic recombination in immunoglobulins
compare to recombination in T-cell receptors?
9.2 How does genetic recombination in immunoglobulins
compare to recombination in T-cell receptors?
9.3 Why is alternative splicing important in antibody production? 9.3 Why is alternative splicing important in antibody production? 9.4 What is the importance of somatic hypermutation and isotype switching in antibody maturation? 9.4 What is the importance of somatic hypermutation and isotype switching in antibody maturation? 9.5 What is the nature of the different antibody isotypes? 9.5 What is the nature of the different antibody isotypes?

3. © 2022 Oxford University Press
What is the structure of
immunoglobulins?
Similarities of immunoglobulins and the T-cell receptor
Both are very specific in the antigen they recognize.
Their diversity is created through somatic recombination.
Essential components of receptor complexes at the surface of
B and T cells required for activation of the specific immune
response.
Variety of functions different from T cell receptors:
Promote B-cell activation after binding antigen.
In soluble form, immunoglobulins bind to antigens, leading
to:
Antigen neutralization (rendering antigen inactive).
Antigen removal.

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Important discoveries
in immunoglobulin
research
•
Immunoglobulins first
described by:
Emil von Behring and
Shibasabura Kitasato in 1890.
They used serum from
animals immunized against
diphtheria to successfully
treat other animals infected
with the same illness.
This suggested that some
component of the serum
conferred immunity on the
animals receiving it.

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Important discoveries in
immunoglobulin
research (cont’d)
•
Biochemist Paul Ehrlich described
the first theoretical structure of
immunoglobulins (1900).
•
This model refined by Gerald
Edelman and Rodney Porter, who
independently confirmed the
structure of immunoglobulins by
determining:
Their primary sequence
(Edelman).
Their sensitivity to proteases
such as papain (Porter).

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How was the protein sequence of an
entire immunoglobulin determined?

By the 1950s, immunoglobulins had been identified as
playing a critical role in a selective immune response:

Variable regions involved in selectivity.

Constant regions were involved in immune
functions.

Known to be made up of light chains and heavy
chains.

Immunoglobulins had been partially sequenced.

Edelman et al. (1969) sequenced the entire
immunoglobulin protein and determined the location of
disulfide bonds formed between the heavy and light
chains.

They used the data to map an immunoglobulin structure
with respect to the interactions of heavy chains and light
chains.

7. © 2022 Oxford University Press
Immunoglobulin
composition
•
To function, immunoglobulins must be able to:
Bind tightly to their cognate antigen.
Link the bound antigen to other effector mechanisms of the
immune system.
•
Each immunoglobulin is composed of four polypeptides:
Two identical heavy chains and two identical light chains.
Each light chain has a variable region and a constant region
Each heavy chain has one variable region and three or four
constant regions.
Chains covalently linked via disulfide bonds between
cysteines.
•
The isotype is dictated by expressi on of a specific heavy chain: ( μ, δ,
γ, ε, or α heavy chain constant region).

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Immunoglobulin
composition: Isotypes
•
Five immunoglobulin isotypes with specific
functions:
IgM
IgD
IgG
IgE
IgA
•
Each isotype shares a common repeating
immunoglobulin domain.

9. © 2022 Oxford University Press
Immunoglobulins:
antigen-binding site
•
Heavy and light chain linked by a disulfide bond
keeps the chains in close proximity.
•
Analogous to the α and β chains of the T-cell
receptor.
•
Interaction of variable regions of heavy and light
chains creates an antigen-binding site.
•
Because there are two heavy and two light chains,
they form two antigen binding sites per
immunoglobulin.
•
A hinge regiongives flexibility to the molecule.

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Immunoglobulins:
papain digest
•
Digestion of purified immunoglobulin with
papain releases two types of fragments •
Fabportion (fragment antigen-binding)
•
Fcportion (fragment crystallizable)
•
This view of an immunoglobulin allows us
to appreciate how a single molecule is
capable of connecting a foreign pathogen
to an immune response.

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B-cell expression of
immunoglobulins
•
Two forms are expressed by a B
cell:
1. A membrane-bound form that
serves as an antigen receptor.
2. A soluble form that targets a
specific foreign antigen.
•
Immunoglobulin diversity is a
critical part of the humoral
adaptive response.
•
Variable regions of heavy and
light chains form antigen-binding
sites for each immunoglobulin.

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Variable Region
Immunoglobulin
structure
•
Each variable region of the heavy
and light chains contains
sequences of amino acids known
as hypervariable regions.
•
Hypervariable regions are three
flexible loops of each variable
region located at the junction sites
of V, D, and J segments.
•
Diversity of hypervariable regions
is driven by both somatic
recombination and junctional
diversity.

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Immunoglobulin
epitopes
Epitope: specific location
or structure where an
immunoglobulin binds.
Usually consists of a short
sequence or structural
feature of the antigen.
Immunoglobulins can bind
any of the four major
macromolecules: proteins,
lipids, carbohydrates, or
nucleic acids.

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Multivalent
antigens
•
Bind multiple copies of the same
immunoglobulin.
•
Occurs because the antigen has
either:
A repeating epitope for
the same immunoglobulin.
Or multiple different
epitopes for different
immunoglobulins from
separate B cell clones.
•
Clustering of immunoglobulins on
an antigen allows for complement
activation and increased
phagocytic efficiency.

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Linear vs conformational
epitopes
•
Epitopes may be recognized as a linear
sequence or as a specific conformational
shape.
•
Linear epitope is made up of macromolecule
residues in sequence; these typically remain
functional when antigen is denatured
because the linear sequence is conserved,
so the epitope is still complete.
•
Conformational epitope is created by the
complete 3D structure of the antigen; these
are typically destroyed when antigen is
denatured because the 3D structure is lost.

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Genetic recombination
in lymphocytes
•
Somatic recombination is responsible for
the diversity of the adaptive immune
system.
•
Genes for heavy and light chains of
immunoglobulins contain many different V,
D, and J segments.
•
Due to the number of segments and
junctional diversity, a very diverse
population of B cells can be produced.
•
Each produces a distinct immunoglobulin
that recognizes a different epitope.

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Similarities with T-cell
receptor recombination
•
Somatic recombination at the immunoglobulin loci occurs in B cells.
•
Rearrangement begins with the heavy chain loci before continuing to
rearrangement events at the light chain loci.
•
Somatic recombination is identical to the process in T cells through the
activity of:
V(D)J recombinase.
RAG1.
RAG2.
Exonucleases.
TdT.

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B cell receptors vs Antibodies
•
B cells express immunoglobulins at the
cell surface.
•
Immunoglobulins at the B-cell surface have
very short cytoplasmic tails.
•
Must interact with cell-surface proteins Ig α
and Igβto drive the signal transduction
events needed for B-cell activation.

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Immunoglobulin
heavy chain
constant region
genes
•
Somatic recombination at the immunoglobulin loci occurs during B-cell
development in the bone marrow, before the B cell is exposed to antigen.
Similar to what happens during development of naïve T cells in the thymus.
•
Recombination of the T-cell receptor lo ci halts after positive and negative
selection is completed in the thymus.
•
However, recombination events can occur after the B cell has finished
development in the bone marrow.
•
B cells leave the bone marrow and undergo class switch recombinationin
the secondary lymphoid after activation. Heavy chain constant regions and
their corresponding isotypes:
μ: IgM
δ: IgD
γ: IgG
ε: IgE
α: IgA

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Naïve B cell repceptor
reminder
•
Naïve B cells leave bone marrow and
travel to secondary lymphoid tissue.
•
Express IgM and IgD at cell surface.
•
These cells enter the secondary tissue to
survey for specific antigen using their B-
cell receptors.

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alternative splicing is
important in
antibody production Naïve B cells only express
immunoglobulins (IgM and
IgD) as membrane proteins at
their cell surface.
During transcription, a long
primary RNA transcript is
produced containing both μ
and δ constant regions.

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B cell activation
makes soluble Igs
Naïve B cells then circulate to secondary lymphoid
tissue to monitor for the antigen that their
immunoglobulin can bind.
B-cell activation triggered by antigen binding
•clonal expansion is induced along with
differentiation into plasma cells.
•including production of soluble
immunoglobulins.
•production of IgM, IgD, and soluble Igs uses
alternative splicingmechanisms.
All isotypes can be produced as both membrane-bound
and soluble proteins.
•Naïve B cells and memory B cells produce
membrane-bound immunoglobulins.
•Terminally differentiated B cells (plasma
cells) produce soluble immunoglobulins.

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9.3 Why is alternative splicing important
in antibody production?
6
Primary RNA transcript for immunoglobulins
•
This primary RNA transcript contains a segment that encodes a large
hydrophobic sequence of amino acids required to span the plasma
membrane.
•
The segment either can be maintained in the mature mRNA or can be
removed during RNA splicing.
•
If the segment is retained, the immunoglobulin anchors in the plasma
membrane as a membrane-bound receptor.
•
If the segment is removed, the protein becomes a soluble
immunoglobulin and is secreted from the cell.

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Naïve vs Activated B cells
•IgM and IgDplay important initial roles in B-cell
function and in humoral adaptive immunity.
•The other immunoglobulin isotypes (IgA, IgG, and
IgE) are also central to humoral adaptive immunity:
IgA, IgG, and IgE are not expressed in naïve B cells.
Only activated B cells that have gone through isotype switching express IgA, IgG,
and IgE.

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Affinity
maturation
A primary immune response that activates B
cells can induce immunoglobulins to undergo
affinity maturation.
A primary immune response that activates B
cells can induce immunoglobulins to undergo
affinity maturation.
Affinity maturation: the process by which
immunoglobulin genes gain higher affinity for
antigen through mutagenesis of the
immunoglobulin gene during B-cell activation.
Affinity maturation: the process by which
immunoglobulin genes gain higher affinity for
antigen through mutagenesis of the
immunoglobulin gene during B-cell activation.
Affinity maturation enhances the
immunoglobulin’s ability to recognize and bind
to antigen quickly and efficiently.
Affinity maturation enhances the
immunoglobulin’s ability to recognize and bind
to antigen quickly and efficiently.
The altered B cell can become activated more
quickly during a secondary adaptive immune
response.
The altered B cell can become activated more
quickly during a secondary adaptive immune
response.

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Somatic
hypermutation
Activation-induced cytidine deaminase (AID)plays a key role
by deaminating the base cytosine, creating the base uracil,
activating DNA repair mechanisms and leading to point
mutations.
After B cell activation: 
some B cells secrete antibodies
identical to original parent B cell.

other B cells engage in affinity
maturation through somatic
hypermutation.
Somatic hypermutation is
concentrated within the variable
regions, leading to more than
1,000,000 different immunoglobulins
Mutation frequency is much higher
than in other cells:
1 in every 10,000 base pairs in
somatic hypermutation vs 1 in
10,000,000,000 base pairs per cell
division.
Produces 1-2 mutations per variable
region per cell division.

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The mechanism by which AID
preferentially mutates the immunoglobulin
genes within B cells is still being debated. Researchers aimed to identify core
sequence elements of the
diversification activator (DIVAC) that
might be responsible for facilitating
targeted mutation of immunoglobulin
genes by AID.
The researchers identified key
regulatory elements that function as
transcriptional enhancers and, more
importantly, as elements responsible
for enhancing the specific targeting of
hypermutation within a B-cell line.

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DNA repair mechanisms in
somatic hypermutation
•
Three possible mechanisms can repair
the cytosine-to-uracil conversion, and
all three can potentially introduce
mutation at the changed base pair:
DNA replication.
Base excision repair.
Mismatch repair.

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Isotype switching
•
Recombination events are not limited to the variable
regions of the heavy and light chain immunoglobulin
loci.
•
Activated B cells can also undergo class switch
recombination.
•
This allows them to produce immunoglobulins with
effector functions suited for the tissue they are
protecting and the pathogen they are combating.

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AID and uracil-DNA glycosylase
activity
•
Isotype switching requires the activities of AID and uracil-
DNA glycosylase.
•
Mechanisms underlying class switch recombination differ
slightly from those driving somatic hypermutation.
•
Located upstream of each of the constant regions
(except for the δ constant region) are switch regions (also
called switch sequences).
•
These contain repetitive target sequences for AID activity.

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Isotype Switching Products
•
Isotype switching is designed to induce the most
efficient immune response possible against a
pathogen.
•
The antibody structure will vary based on the heavy
chain constant region loci and its expression.
•
Antibody effector functions include:
Neutralization.
Opsonization.
Activation of complement pathways and/or
innate immune cells.

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IgM
•
The IgM isotype is co-expressed with IgD in naïve
B cells.
•
Soluble pentameric form consisting of five
monomers interacting with the soluble J chain
protein.
•
High avidity(combined strength of bond affinities)
for antigen.
•
Binding affinity of antigen-binding sites is lower
because IgM locus does not undergo somatic
hypermutation.
•
Involved in antigen neutralization.
•
Serves as complement activation trigger (classical
pathway).
•
Structure of pentameric IgM sterically blocks the Fc
components of each monomeric immunoglobulin.
•
IgM therefore cannot serve as an opsonin for
phagocytic cells and other innate immune cells.

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IgD
•
Expressed by naïve B cells along with IgM.
•
Involved in B-cell activation.
•
Not highly expressed in soluble form.
•
Plays a role in mast cell and basophil activation similar to
IgE.

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IgA
•
Involved in mucosal surface protection.
•
Found in both monomeric and dimeric
forms (held together by J chain protein).
•
Serves as neutralizing antibody.

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IgG
•
Most abundant immunoglobulin in serum.
•
Four subclasses differing in the heavy chain constant
regions and the length of the hinge regions.
•
Serves as neutralizing and opsonizing antibody.
•
Can also substitute for C-reactive protein in the classical
complement pathway.

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IgE
•
Bridge between innate and adaptive immune response.
•
Acts on inhaled or ingested pathogens, including
parasites.
•
Associates with granulocytes such as mast cells,
basophils, and eosinophils through interaction of its
heavy chain constant region with Fc receptors on the
surface of these cells.