Chapter 4 – Antibody Structure and the Generation of B-Cell Diversity
I.
II.
III.
IV.
V.
Antibodies can recognize all types of biological macromolecules, but in practice, proteins and
carbohydrates are the antigens most commonly encountered.
Collectively, antibodies are diverse in their antigen-binding specificities – the total number that
can be made (antibody repertoire) might be as high as 1016; the number of B cells limits the
actual repertoire to 109
Before encountering antigen, mature B cells only express membrane-bound immunoglobulin
that serves as the cell receptor for antigen; when an antigen binds to this receptor, B cells are
stimulated to proliferate and differentiate into plasma cells, which then secrete large amounts
of antibodies with specificity for that membrane-bound immunoglobulin.
The structural basis of antibody diversity
a. Antigen binding/interaction with other immune system cells are carried out by different
part of the antibody molecule
i. One part is highly variable (amino acid sequence differs considerably from
antibody to antibody)
1. Contains sit of antigen binding and confers specificity
ii. One part is constant and interacts with other immune-system components
b. Immunoglobulins are split into 5 classes (isotypes) – IgA, IgD, IgE, IgG and IgM
i. Distinguished based on structure differences in the constant part of the
molecule; different effector functions
Antibodies are composed of polypeptides with variable and constant regions
a. Antibodies are glycoproteins built from a basic unit of four polypeptide chains (two
heavy [H] chains and two identical, smaller light [L] chains) – assembled to look like the
letter Y.
i. Each arm is made of a complete light chain,
paired with the amino terminal part of the
heavy chain (chains are covalently linked by
a disulfide bond)
b. Variability is the reason for great diversity of antigen-binding
specificities among antibodies, because the paired variable
(V) regions of a heavy and light chain form the antigenbinding site
i. Each antibody has two identical antigen binding sites
(one at the end of each arm)
c. In IgG, a relatively unstructured portion in the middle of the
heavy chain forms a flexible hinge region, at which it can be
cleaved to produce defined antibody fragments
i. Digestion with the plant protein papain produces 3
fragments –correspond to two arms and a stem
1. Arm fragments – FAB (FRAGMENT ANTIGEN BINDING)
2. Stem fragment – Fc (FRAGMENT CRYSTALLIZABLE)
The structures of the human immunoglobulin classes
Note differences in H chain C-regions, location of disulfide bonds linking the chains, and presence of a hinge region in IgG, IgA,
and IgD. All isotypes ccur as monomers in their membrane bound form; in their soluble, secreted form IgD, IgE and IgG are
always monomers, IgA forms monomers and dimers, and IgM forms only pentamers.
VI.
VII.
d. Differences in the H chain C-regions define the five isoptypes of immunoglobulin
i. Their heavy chains are denoted by ϒ (IgG), µ (IgM), δ (IgD), α (IgA), ε (IgE)
e. The L chain only has two isotype classes –κ and λ
i. Light chains of both isotypes are found associated with all H chain isotypes; each
antibody contains either the κ OR λ light chain isotype, NOT BOTH.
Immunoglobulin chains are folded into compact and stable protein domains
a. The V region at the amino-terminal end of each heavy or light chain is composed of a
single variable domain (V domain) – VH in the heavy chain and VL in the light chain –
these domains together form the antigen binding site
i. The other domains have little/no sequence diversity C domains make up the C
regions
b. The structure of a single immunoglobulin domain can be compared to a bulging
sandwich in which two β sheets are held together by strong hydrophobic interactions
between their constituent amino acid side chains; structure stabilized by disulfide bonds
between the two sheets.
An antigen binding site is formed from the hypervariable regions of a heavy chain V domain and
a light chain V domain
a. Comparison of V domains of heavy and light chains from different antibody molecules
show the differences in amino acid sequences are concentrated within hypervariable
regions, which are flanked by much less variable framework regions
i. Three HV regions are found in each V domain
ii. Framework regions correspond to the β strands and the remaining loops
b. The pairing of heavy and light chain in antibodies brings together the hypervariable
loops from each V domain to create the composite hypervariable surface (forms antigen
binding site at the tip of each FAB arm)
c. These hypervariable loops are also called complementarity-determining regions (CDR)
VIII.
IX.
Antigen-binding sites vary in shape and physical properties
a. Antigenic determinant (epitope) is the part of antigen to which antibody binds
i. Are usually either carbohydrate or protein, because the surface molecules of
pathogens are commonly glycoproteins, polysaccharides, glycolipids, and
peptidoglycans
1. complex macromolecules such as these contain multiple epitopes, each
of which can be bound to different antibody (multivalent)
b. Antigen binding site of antibodies vary according to size and shape of epitope they
recognize
i. Epitopes can bind to pockets, grooves, extended surfaces, or knobs in antigenbinding sites
1. Linear epitopes-antibody binds to parts of the molecule that are
adjacent in the linear sequence
2. Conformational (discontinuous) epitopes – formed by parts of a
protein that are separated in the amino acid sequence, but are brought
together in the folded protein.
ii. The binding of antigen to antibodies is based solely on non-covalent forces –
electrostatic forces, hydrogen bonds, van der Waals forces, and hydrophobic
interactions
1. Binding caused by van der Waals forces and hydrophobic interactions is
complemented by the formation of electrostatic interactions and
hydrogen bonds between particular chemical groups on the antigen and
particular amino acid residues of the antibody
iii. Some antibodies have been found to catalyze reactions involving the antigen
that they bind – catalytic antibodies
Monoclonal antibodies are produced from a clone of anti-body producing cells
a. The traditional method for making antibodies of desired specificity is to immunize with
the appropriate antigen and the prepare antisera from their blood.
b. More modern method – monoclonal antibodies
i. B cells are isolated from immunized animals and
immortalized by fusion with a tumor cell to form
hybridoma cell lines that will grow and produce
antibodies indefinitely. Individual hybridoma cells
are then separated, and cells making antibodies of
the desired specificity are identified and selected for
further propagation.
c. Monoclonal antibodies are used in therapy and diagnosis
i. First successful – mouse monoclonal antibody specific for
CD3 antigen on human T cells; block T-cell response and
prevent imminent T cell mediated rejection of transplanted kidneys
1. Limited because human immune system perceives mouse antibody as
foreign; chimeric monoclonal antibodies reduce this problem (combine
mouse V regions with human C regions)
2. Another approach to reducing the tendency for useful mouse
monoclonal antibodies to provoke an immune response is to humanize
them.
a. Sequences encoding CDR loops of mouse monoclonal antibody
H and L chain are used to replace corresponding CDR sequences
of a human immunoglobulin.
X.
Generation of immunoglobulin diversity in B cells before encounter with
antigen
a. For an immunoglobulin gene to be expressed, individual gene segments must be
rearranged to assemble a functional gene – occurs only in developing B cells
i. Once complete, heavy and light chains can be produced and membrane bound
immunoglobulin appears on B cell surface allowing the cell to recognize and
response to antigen
b. In humans, immunoglobulin genes are found at thee chromosomal locations
i. Heavy chain locus on chromosome 14, the κ light chain locus on chromosome 2
and the λ light chain locus on chromosome 22.
c. Gene segments encoding leader peptides and the C regions consist of exons and introns
i. In contrast, V regions encoded by two VL or three VH gene segments require
rearrangement to produce an exon that can be transcribed
1. The two types of gene segments that encode the light chain V region are
called variable (V) and joining (J) segments
2. the heavy chain locus includes an additional set of diversity (D) gene
segments that lie between the V and J segments
d. Random recombination of gene segments produce diversity in the antigen-binding sites
of immunoglobulins
i. During B cell development, arrays of V, D and J segments are cut and spliced by
DNA recombination (somatic recombination)
1. Single gene segment is brought together to form a DNA sequence
encoding the V region of an immunoglobulin chain
2. For light chains, a single recombination occurs between a VL and JL
segment; for heavy chains, two recombinations are needed – first to
join a D and JH segment and then to joined the combined DJ segment to
a VH segment.
a. RANDOMLY SELECTED – numerous different combinations of V,
D, and J segments are possible
3. Somatic recombination is carried out by enzymes that cut and rejoin the
DNA; exploits mechanisms more universally used by cells for DNA
recombination and repair
a. Recombination of V, D, and J segments is directed by
recombination signal sequences (RSS) – which flank the 3’ side
of the V segment, both sides of the D segment and the 5’ side of
the J segment
i. Two types – 1. a heptamer sequence [7 base pairs:
CACAGTG] and a nonamer [9 base pairs: ACAAAAACC]
separated by a 12 base pair spacer; 2. The heptamer
and the nonamer sequences separated by a 23 base
pair spacer
ii. Recombination in the heavy chain CANNOT join VH
directly to JH without the involvement of DH, because
the VH and JH segments are flanked by the same type of
RSS.
e. Recombination enzymes produce additional diversity in the antigen-binding site
i. V(D)J recombinase – set of enzymes needed to recombine the V, D, and J
segments
1. Two of the component proteins are made only in lymphocytes –
specified by recombination-activating genes (RAG-1 and RAG-2)
2. Other components are present in all nucleated cells and have activities
to repair double-stranded DNA, bend DNA, or modify ends of broken
strands (DNA ligase IV, DNA-dependent protein kinase, the nuclease
Artemis, and the Ku protein associated with DNA-PK)
3. RAG-1 and RAG-2 proteins interact with each other and with other
proteins to form the RAG complex
a. One RAG complex binds to one type of RSS and another
complex binds to the other type of RSS
b. Interaction between RAG complexes aligns the two RSSs and
cleaves the DNA at the ends of the immunoglobulin gene
segment in such as a way as to create a hairpin at the end of
each segment and a clean break at the end of the two heptamer
sequences
i. The DNA molecules are held in
place by the RAG complexes
while the broken ends are
rejoined by DNA repair
enzymes in a process called
nonhomologous end-joining
The generation of junctional diversity during gene rearrangement
(forms a coding joint and a
signal joint)
c. Signal joint – joined ends of the removed DNA; Coding joint –
joined ends of two gene segments
4. The enzymes that open hairpins and form the coding joint introduce
additional diversity to the third hypervariable region (CDR3) of
immunoglobulin heavy and light chains
a. The RAG complex generates palindromic “P” nucleotides, which
allows the opened hairpins to be variably modified by
exonucleases that remove germline encoded nucleotides and by
the enzyme terminal deoxynucleotidyl transferase (TdT), which
randomly adds nucleotides
i. Added nucleotides are called N nucleotides because
they are non-templated (not encoded) in germline DNA.
b. Once single-strand tails of two gene segments are able to pair,
the gaps are filled with complementary nucleotides to complete
the coding joint.
c. Contribution of P nucleotides and N nucleotides to the resulting
amino acid sequence diversity in CDR3 – junctional diversity
i. Important source of immunoglobulin variability
ii. The third hypervariable region of the light-chain V
domain is encoded by the junction between the V and J
segments; the third hypervariable region of the heavy
chain V domain is formed by the D segment and its
junctions with the rearranged V and J segment
Rearrangement of V, D, and J segments produces a functional heavy-chain gene
f.
Developing and naïve B cells use alternative mRNA splicing to make IgM and IgD
i. Naïve B cells – express both IgM and IgD on their surface ; have yet to
encounter antigen (IgM and IgD are the only isotypes that can be produced
simultaneously by a B cell)
ii. Rearrangement of the V, D, and J segments of the heavy-chain locus that occurs
during B-cell development brings a gene promoter and enhancer into closer
juxtaposition, enabling transcription
1. Resulting mRNA transcript is spliced, processed and translated to give a
heavy-chain protein
a. The exons encoding leader peptide and the V region are on the
5’ side (upstream) of the DNA encoding the nine different C
regions
b. Closest to the rearranged V region is the µ-gene, followed by
the δ-gene
2. In mature naïve B cells, transcription of the heavy chain starts upstream
of the exons encoding the leader peptide and the V region, continues
through the µ and δ C genes, and terminates downstream of the δ gene,
before the ϒ3 C gene.
a. This long primary RNA transcript is then spliced and processed
in two ways – one yields mRNA for the µ heavy chain and one
yields mRNA for the δ heavy chain
i. In making the µ-chain mRNA, the entire δ gene RNA is
removed along with the introns from the µ gene.
ii. In making the δ-chain mRNA, the entire µ gene RNA is
removed as well as the δ-gene introns.
g. Each B cell produces immunoglobulin of a single antigen specificity
i. Allelic exclusion - the process in a developing B cell of immunoglobulin-gene
rearrangement in which the process is tightly controlled so that only one heavy
chain and one light chain are expressed
1. Even though every B cell has two copies of the heavy chain locus and
two of each light chain locus, only ONE heavy-chain locus and ONE light
chain locus are rearranged to produce functional genes
ii. Because an antigen-binding site is formed by the association of a heavy chain
and a light chain, the combinatorial association makes an important
contribution to the overall diversity of immunoglobulins
iii. Monospecific – encounter with a given pathogen engages a subset of B cells
that will make antibodies that bind only to the pathogen
iv. Because the DNA sequence of expressed immunoglobulin genes varies from one
clone of B cells to the next can be used to detect large clonal populations of
cancer cells in patients with B-cell lymphoma or leukemia
h. Immunoglobulin is first made in a membrane-bound form that is present on the B cell
surface
i. When a B cell first makes IgM and IgD, the heavy chains have a hydrophobic
sequence near the carboxy terminus by which the immunoglobulins associate
with cell membranes
ii. By themselves, these immunoglobulin molecules cannot be transported to the
cell surface.
1. For this to happen, the must associate with two transmembrane
proteins Igα and Igβ
a. Proteins are invariant in sequence, and travel to the B cell
surface in a complex with the immunoglobulin molecule
b. At the surface, this complex forms the B cell receptor for
antigen
XI.
Diversification of antibodies after B cells encounter antigen
a. All the isotypes of immunoglobulin can be made in two forms:
i. one that is bound to the cell membrane and serves as the B cell receptor for
antigen
ii. one, the antibody, that is secreted to bind to antigen and aid its destruction
b. During differentiation to antibody-secreting plasma cells, B cells change from making
membrane-bound form to making the secreted form; plasma cells ONLY make secreted
antibody
c. The difference between membrane-bound and secreted immunoglobulin lies at the
carboxy terminus of the heavy chain:
i. Membrane-bound: has a hydrophobic anchor sequence that is inserted into the
membrane
1. The hydrophobic anchor of the membrane-associated µ chain is
encoded by two small, separate exons downstream.
2. The splicing to give membrane-bound µ chain: alternative splicing in the
exon encoding the fourth C-region domain removes the sequence
encoding the hydrophillic, whereas the exons encoding the hydrophobic
carboxy terminus are retained and incorporated into the mRNA when
the introns are spliced out.
ii. Secreted: has a hydrophilic sequence
1. The hydrophilic caryboxy terminus of the secreted µ chain is encoded at
the 3’ end of the exon encoding the fourth C-region domain
2. The splicing to give secreted µ chain: the sequence encoding the
hydrophilic carboxy terminus is retained and the sequences 3’ of that,
including the exons encoding the hydrophobic membrane anchor, are
discarded.
iii. Difference is determined by different patterns of RNA splicing and processing of
the same primary RNA transcript; involves NO rearrangement of underlying
genomic DNA.
d. Rearranged V-region sequences are further diversified by somatic hypermutation
i. Diversity generated during gene rearrangement is concentrated in the third CDR
of the VH and VL regions. (somatic hypermutation)
1. Randomly introduces single nucleotide substitutions (point mutations)
at a high rate throughout the rearranged V regions of the heavy and
light chain genes
ii. Somatic mutation is dependent on the enzyme activation-induced cytidine
deaminase (AID) – which is made ONLY by proliferating B cells
1. Converts cytosine in single-stranded DNA to uracil, a normal component
of RNA but not DNA
2. Other enzymes, that are not specific to B cells but are components of
general pathways of DNA repair and modification, can then act to
convert the uracil to any one of the four bases of normal DNA
iii. Somatic hypermutation gives rise to B cells bearing mutant immunoglobulin
molecules on their surface
1. Some mutant immunoglobulin molecules have substitutions in the
antigen-binding site that increase its affinity for the antigen
iv. As the adaptive immune response proceeds, antibodies of progressively higher
affinity for the infecting pathogens are produced – a phenomenon called
affinity maturation
1. Process of evolution in which variant immunoglobulins generated in a
random manner are subjected to selection for improved binding to a
pathogen
e. Isotype switching produces immunoglobulins with different C regions but identical
antigen specificities
i. IgM is the first antibody made in primary immune response
1. Membrane-bound IgM of the B cell receptor is monomeric, whereas
secreted IgM consists of a circular pentamer of the Y shaped
immunoglobulin monomers
2. IgM binds strongly to the surface of pathogens with multiple repetitive
epitopes, but is limited in effector mechanisms that it uses to clear
antigen from the body
ii. Isotype switching (class switching) – a further DNA recombination event
enables the rearranged V region coding sequence to be used with other heavy
chain C-genes.
1. Is dependent on AID and similarly only occurs in B cells proliferating in
response to antigen
2. Accomplished by recombination within the cluster of C genes that
previously expressed C gene and brings a different one into
juxtaposition with the assembled V-region sequence
a. Thus, antigen specificity remains unchanged; isotype changes
3. Flanking the 5’ side of each C gene, are highly repetitive sequences that
mediate recombination – switch regions (except on the δ gene)
Isotype switching involves recombination between specific switch regions
Repetitive DNA sequences are found to the 5’ side of each heavy chain C-gene (except δ gene). Immunoglobulin isotype
switching occurs by recombination between the switch regions (S), with deletion of the intervening DNA. The switch regions are
targeted by AID, which leads to nicks being made in both strands of DNA. The nicks facilitate recombination between the switch
regions, which leads to excision of intervening DNA as a non-functional circle of DNA and brings the rearranged VDJ segments
into juxtaposition with a different C gene. The first switch a clone of B cells makes is from the µ isotype to another isotype. A
switch from µ to the ϒ1 isotype is shown here. Further switching to other isotypes can take place subsequently.
f.
iii. In patients that lack a functional AID gene cannot undergo somatic
hypermutation or isotype switching; only produce low-affinity IgM (Hyper IgM
Immunodeficiency)
1. Main consequence is susceptibility to infection by pyogenic bacteria,
particularly in sinuses, ears and lungs
Antibodies with different C regions have different effector functions
i. The certain classes of immunoglobulin are further divided into subclasses, which
differ in both nomenclature and properties between species.
1. IgA IgA1 and IgA2
;
IgG IgG1, IgG2, IgG3, and IgG4
(numbered according to relative number in plasma)
2. Heavy chains of IgA subclasses are designated α1 and α2 and the heavy
regions of IgG subclasses are ϒ1, ϒ2, ϒ3, and ϒ4
ii. Neutralizing Antibodies – directly inactivate pathogens/toxins and prevent it
from interacting with human cells
XII.
XIII.
XIV.
XV.
1. Bind to a site on pathogen that is normally used to infect cells ;
opsonization (coating pathogens with immune system protein)
a. Opsonized pathogen are more efficiently ingested by
phagocytes, which have receptors for the Fc region of some
antibodies and for certain complement proteins
iii. Overall strength of binding at multiple sites – avidity
iv. Strength of binding to a single site – affinity
v. By isotype switching, different effector functions can be brought into play
while preserving antigen specificity: synthesis of IgM gives way to synthesis of
IgG
IgM is made primarily by plasma cells resident in lymph nodes, spleen and bone marrow;
circulates in blood and lymph
IgG is the most abundant antibody in internal fluids
a. Made principally in lymph nodes, spleen and bone marrow ; circulates in lymph and
blood.
b. Structure is smaller and more flexible than IgM, giving it easier access to antigens in the
extracellular spaces of damaged and infected tissues
c. Can be transferred across the placenta to provide the fetus with protective antibodies
from the mother, in advance of possible infection
Monomeric IgA is made by plasma cells in lymph nodes, spleen and bone marrow and is
secreted into the blood stream
a. Can also be made as a dimer
i. Dimeric IgA is principally found in lymphoid tissues underlying mucosal surfaces
and is the antibody secreted into the lumen of the gut; is also the main antibody
in other secretions including milk, saliva, sweat and tears
ii. Some effector function is directed against resident microogansisms that
colonize mucosal surfaces, keeping their population in check
IgE is highly specialized toward recruiting the effector functions of mast cells in epithelium,
activated eosinophils present at mucosal surfaces, and basophils in blood.