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Antibodies 1: Structure of Antibodies
5-3
Antibodies contain an antigen-binding region and a region that
connects to effector mechanisms
Antibodies, the first recognized members of the immunoglobulin superfamily (see Chapter 2),
are heteromeric proteins composed of two types of chains, both of which contain multiple Ig
domains. Unlike those of the TCR however the two chains are of very different sizes: one,
known as the light chain, is similar in size to a TCR chain; the other is known as the heavy
chain and is much larger. It is the heavy chain that has a transmembrane region and in the
membrane-bound receptor form of the molecule anchors the molecule in the membrane; in
the secreted form—the antibody—the heavy chain is responsible for engaging with the cells
and molecules that destroy the microorganisms to which the antibody binds. The basic design
of antibodies is illustrated for IgG in Figure 5-1.1. The structure contains two identical heavy
chains and two identical light chains: each light chain associates with a heavy chain creating
two identical antigen-binding units. The two heavy chains also pair with each other. The
chains are linked together by disulfide bonds.
Figure 5-1.1 Basic structure of antibodies
IgG consists of paired heavy and light chains in
a H2L2 structure. The names of individual
domains are indicated on the right half of the
molecule (a). Disulfide bonds (represented as
yellow lines) covalently link light chains to
heavy chains and also heavy chains to each
other in the hinge region (the number of
disulfide bonds varies with the different
subclasses of IgG, for example, IgG1, IgG2,
IgG3, or IgG4). The sites of papain and pepsin
cleavage are shown. Papain cleavage gives rise
to two Fab fragments and one Fc fragment.
Pepsin cleavage gives rise to primarily a single
F(ab’)2 fragment; the Fc fragment is cleaved
further by pepsin. The antigen binds to a site
contributed by the VH and VL domains, whereas
C1q and Fc receptor binding sites are in the Fc
part of the molecule. (b) Ribbon diagram of
IgG. Note that the domains are not arranged in
a strictly linear array, but rather there are
generally bends between adjacent domains. In
the Fc region, this is in part due to
carbohydrate attached to CH2, which sterically
separates these domains, in contrast to the CH3
domains, which make close contact with one
another.
Definitions
complementarity-determining region: region of antibody structure that binds to antigen, made up of the
hypervariable loops at one end of the V domains of the
heavy and light chains.
constant domain: Ig domains that are the same for all
antibodies of a particular isotype.
Fab fragment: fragment of an antibody generated by
limited digestion with the protease papain. The Fab
fragment contains the light chain plus the V and CH1
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The specificity of antibodies derives from the N-terminal Ig domains of each chain, which pair
with each other. These variable (V) domains, like those of TCR chains, can have many different amino acid sequences and are generated during B cell development from multiple gene
segments along with variation introduced at the joints, a process called V(D)J recombination,
by essentially the same mechanism as for V domains of TCR. This process is outlined in Figure
5-1.2 and will be discussed in detail in Chapter 7. Antigen-binding specificity derives from the
loops at one end of the V domains of both the light chain and the heavy chain (Figure 5-1.3),
and these loops contain most of the variability from one antibody to the next. Like those of
the TCR, they are known as hypervariable regions. As they form the basis for binding to antigen, a process that is in part based on shape complementarity, they are also referred to as complementarity-determining regions.
The structure of the complementarity-determining regions can vary considerably. The antigenbinding site of some antibodies is a relatively flat surface which is good for binding to native
protein antigens, whereas for other antibodies it can contain a deep pocket or a cleft, in which
small organic molecules, oligosaccharides, and so on, can bind.
The rest of the antibody molecule has much less variation than the V domains, and therefore
the Ig domains that comprise these parts of the molecule are called constant (C) domains.
(a) antigen binding
(b)
VH
hinge
CH1
VL
Fab
CL
Fc
(binding
of FcRs)
CH2
papain
pepsin
CH3
domain of the heavy chain, and represents a monomeric antigen-binding fragment without effector functions.
Fc fragment: fragment of an antibody generated by
limited digestion with papain.The Fc fragment contains
only the CH2 and CH3 domains of the heavy chains. Fc
stands for fragment crystallizable, reflecting the homogeneity of the Fc regions by comparison with the Fab
regions of polyclonal antibodies, which allows Fc
regions to be crystallized.
hinge region: a stretch of amino acids between CH2
and CH3 with flexibility, allowing the antigen binding
sites mobility relative to one another.
hypervariable region: regions of the V domain of the
heavy or light chain with the greatest variability. These
correspond to loops in the Ig domain structure and
form the antigen-binding site.
immunoglobulin superfamily: a family of proteins
each of which contains at least one Ig domain as part of
its structure.
variable domain: The N-terminal Ig domains of the
heavy and light chains where the variability is greatest
©2003 New Science Press Ltd new-science-press.com
Figure 5-1.2 Functional immunoglobulin genes of B cells are generated by somatic recombination
between gene segments Shown is a representation of the IgH locus. At the top is the configuration of
the germ-line chromosomal DNA. In B cell precursors, one DH gene segment is moved next to one JH
segment by deletion of the intervening DNA and then one VH gene segment is juxtaposed to the DJ
combination, again by deleting intervening DNA, resulting in a single exon composed of V, D and J
segments (middle). The D and J segments contribute amino acid sequence to the third loop in the Ig
domain, which corresponds to the third complementarity-determining region of the heavy chain (CDR3;
also called hypervariable region 3; see next section). If the open reading frame of the D and J segments
is maintained, then this is a functional rearrangement that can encode an Ig heavy chain. Transcription of
the rearranged Ig gene allows production of functional mRNA after RNA splicing by the normal
mechanisms (bottom). An analogous process occurs at the light chain loci, except that there are only V
and J regions. These genetic mechanisms allow for encoding of approximately 107 different specificities
by relatively little genetic information. In addition, there is variability in the exact sites of recombination
between the segments and additional bases can be inserted or deleted at the junctions, creating
additional junctional diversity in the CDR3 loops. These features are described in more detail in Chapter 7.
germline DNA
VH1 VH2
VH65
DH1DH2 DH27 JH1–JH6
Cµ
3′
lymphocyte DNA
VH1 VH2 JH4–JH6
Cµ
3′
DH2
mRNA encoding assembled receptor chain
AAA
Light chains have one C domain, whereas heavy chains have either three or four, depending
on the type of heavy chain. It is the C domains of the heavy chains that are responsible for the
effector functions of antibodies.
The structure of antibodies was originally deduced in part from studies of the fragments of
antibodies that could be generated by treatment with proteases such as papain and pepsin, and
the mode of cleavage by these enzymes and the fragments they generate are still instructive.
Limited proteolysis cleaves antibodies between Ig domains and the most sensitive site in IgG
molecules is between the first and second constant domains (numbered from the N-terminus).
Between these domains is an extended sequence of amino acids called the hinge region. The
hinge has a flexibility that allows the two antigen-binding sites to have considerable mobility
relative to one-another, facilitating the binding of antibodies to antigens in fixed positions on
virions, bacterial surfaces, and so on. Cleavage of the hinge with papain generates two fragments, one containing the light chain bound to the V and CH1 domains of the heavy chain,
called the Fab fragment, and a second containing the CH2 and CH3 domains of the two heavy
chains. This essentially represents the cleavage of the molecule into its two functional parts: the
antigen-binding regions comprising the Fab fragments, and the effector region, comprising
the Fc fragment. (Fc stands for fragment crystallizable, and reflects the fact that in early studies of antibodies, only the structurally uniform Fc regions of antibodies could be crystallized:
because there was at that time no way of obtaining large quantities of antibody with the same
V region, Fab fragments were always mixtures and were impossible to crystallize.) Pepsin
cleaves at a slightly different site that leaves the two Fab fragments (called Fab’, since they are
slightly different from papain-generated Fab fragments) connected by a disulfide bond. Thus,
pepsin treatment generates F(ab’)2 fragments. Fab and F(ab’) 2 fragments can be used to block
recognition experimentally and therapeutically, without incurring effector actions that may
have unwanted effects.
Figure 5-1.3 Hypervariable regions of the V domains The variable domains of Ig heavy and light
chains have much of their variability clustered in three regions each, referred to as hypervariable regions
1, 2 and 3. These hypervariable regions (shown in green) correspond to loops in the Ig domains that are
at the end of the molecule. These six loops are all located near one another and comprise the antigenbinding site. For this reason, these loops are also called the complementarity determining regions
(CDRs).
and antigen binding function is contained.
The Adaptive Immune Response II: B Cells and Humoral Immunity Chapter 5
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