Last Update: 2 November 2017

ZOO
Part I
M - 04
Basic structure and functions of immunoglobulin M

Q. Prove that antibodies belong to gammaglobulin fraction of serum.

Classic experiment by Tiselius and Kabat 1939:
Immunized rabbits with egg white (ovalbumin). Collected
serum and divided into to parts.
Serum #1 ran on an electrophoresis gel and separated by
charge. Serum #2 was first reacted with ovalbumin and
the precipitate removed. This was then run on a gel.
Below are the results of this experiment:

Serum #1 had 4 peaks. But in serum #2 the gamma peak

was gone because it had bound to the ovalbumin and
precipitated out. This was the immune reactive fraction
or globulin thus the name gamma-globulin or immunoglobulin

Q. What is immunoglobulin? What is the Basic Structure of Antibody? How their structures were
determined? What are different types of the antibodies? Discuss their functions.
Immunoglobulins (Ig) - Glycoprotein molecules which are produced by plasma cells in response to an
immunogen and which function as antibodies. The immunoglobulins derive their name from the finding that when
antibody-containing serum is place in an electrical field the antibodies, which were responsible for immunity,
migrated with the globular proteins
(Figure ).
Antibodies are either membrane bound or secreted. The membrane bound antibody serves as part of the B cell
receptor (BCR) and confers B cell antigen-specificity.
The basic structure of antibody is shared by all antibodies. It consists of 4 chains. These chains are held together by
disulfide bonds, salt linkages and hydrogen bonds. The four chains consist of:
2 light chains (25,000 Da each)
2 heavy chains (50,000 Da each)
150,000 Da total molecular weight

Light chain (L) is bound to the heavy chain by disulfide

bonds and the 2 heavy chains (H) are bound together by
disulfide bonds.
The first 100-110 amino acids of the amino-terminus
(NH3+)of the H and L chains is called the variable region (VL
and VH). It is called this because when scientist sequenced
several individuals serum antibodies, the most variability in
amino acid sequence was found in this area of both chains.

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The variable region is the area that confers the antibody specificity for a particular antigenic epitope. This is because
the variability allows for complimentarity between the antibody and antigen.VH and VL can be divided into two
regions. The hypervariable region is the region that was found to have the highest amount of variability in amino
acid sequence. This is only about 15-20% of the total amino acids that make up the variable region. The
hypervariable region is also called the complementarity determining region (CDR). V H and VL chains have 3 CDRs
each.
STRUCTURE OF THE VARIABLE REGION
A. Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons of the amino acid sequences of the variable regions of Ig's show that most of the variability resides in
three regions called the hypervariable regions or the complementarity determining regions as illustrated in Figure 3.
Antibodies with different specificities (i.e. different combining sites) have different CDR's while antibodies of the
exact same specificity have identical CDR's (i.e. CDR --> Ab Combing site). CDR's are found in both the H and the
L chains.
B. Framework regions
The regions between the CDR's in the variable region are called the framework regions (FR) (Figure ). Based on
similarities and differences in the framework regions the immunoglobulin heavy and light chain variable regions can
be divided into groups and subgroups. These represent the products of different variable region genes.
The rest of the VH and VL region is not as variable and is known as the framework region. Based on amino acid
sequencing data, the remaining regions of the H and L chain are called the constant regions. The L chain has only 1
constant region. The H chain has 3 or 4 constant regions depending on the antibody. See picture above.
Detail structure:
Light chains: Sequencing of the serum antibody of several different people showed different things about the light
chains. First, it defined the variable regions as described above. Second it showed that there were two main types of
constant regions (CL). These were called kappa (k) and lambda (l). In humans, 60% of their light chains are kappa
and 40% are lambda. In mice 95% of the light chains have the kappa sequence and only 5% are lambda. Any given
antibody can only have kappa or lambda light chains but not both.
Heavy chains: Again, as stated above, the amino acid sequencing of the heavy chains showed a variable region
similar to that found in light chains. The rest of the heavy chain is considered constant (C H). There were 5 common
sequences found in the heavy chain constant regions. These were called isotypes or classes of antibodies and are
IgG, IgD, IgE, IgA and IgM. Therefore, it is the constant region of the antibody heavy chain that determines that
antibody's isotype or class.

Disulfide bonds
1. Inter-chain - The heavy and light chains and the two heavy chains are held together by
inter-chain disulfide bonds and by non-covalent interactions The number of interchaindisulfide bonds varies among
different immunoglobulin molecules.
2. Intra-chain - Within each of the polypeptide
chains there are also intra-chain disulfide bonds.
Variable (V) and Constant (C) Regions -
After the amino acid sequences of many different
heavy chains and light chains were compared, it
became clear that both the heavy and light chain
could be divided into two regions based on
variability in the amino acid sequences.
1. Light Chain - VL (110 aa) and CL (110 aa)
2. Heavy Chain - VH (110 aa) and CH (330-440 aa)
Domains - 3D images of the immunoglobulin
molecule shows that it is not straight as depicted
in Figure 2. Rather, it is folded into globular
regions each of which contains an intra-chain
disulfide bond. These regions are called domains.
1. Light Chain Domains - VL and CL
2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)
Oligosaccharides - Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some
cases carbohydrates may also be attached at other locations.IgG and IgA have subclasses. IgG: IgG 1, IgG2, IgG3
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and IgG4. IgA: IgA1 and IgA2.CH regions are divided into either 3 or 4 different CH domains.Like variable regions,
the constant region and domains have specific functions.
CH1 and CL:
extends the Fab arms- facilitates antigen/antibody interaction
holds VH and VL chains together
contributes to antibody diversity allowing for more random associations between VH and VL than if only VH and VL
were involved.
Hinge region:
IgG, IgA and IgD have heavy chains that contain a hinge region. This is a proline rich domain that is found
between CH1 and CH2 domains of these classes of antibody. This region allows for greater flexibility of the Fab.
This allows for various angles to be achieved and increases antigen binding.
Other constant region domains:
Below is a list of antibodies and the constant region domains that they have:
The CH2 domain of IgG and the CH3 domain of IgM is important for IgG, IgA, IgD IgE, IgM
complement activation. Complement is a system of serum proteins that
serve to perforate cell membranes. (we will worry about the specifics later) CH1 CH1
The CH3 domain of IgG, IgA and IgD and the CH4 domains of IgE and
Hinge region CH2
IgM determine whether an antibody is secreted or membrane bound. The
carboxy-terminal region of these domains contain three regions if CH2 CH3
membrand bound
an extracellular spacer of about 26 amino acids (hydrophillic) CH3 CH4
a transmembrane domain a short cytoplasmic tail.

Deducing antibody structure:

Several experiments were conducted to understand how
the H and L chains interact with one another. These were a
series of enzymatic digestions and chemical treatments.
Porter and Engelman (1950s and 1960s) won the Nobel
prize for their work on antibody structure.
Papain: short digestions and electrophoresis led to the
discovery of two fragments. One fragment bound antigen
and was called Fab the other fragment crystallized when
stored at 4 C and was called Fc. Because of this result, the
scientist knew that there has two functional parts of the
antibody.
Pepsin: A scientist called Nisonoff digested antibody with
a different enzyme called pepsin. This digestion (following
electrophoresis) led to one large molecule consisting of
two Fab fragments together and many small fragments of
the Fc region.

Mercaptoethanol: Mercaptoethanol is a reducing agent

and therefore breaks disulfide bonds. The scientists were
able to determine that there were heavy and light chains
because incubation with mercaptoethanol resulted in only
two types of molecules based on molecular weight. A
heavy chain (50,000 Da) and a light chain (25,000 Da).
The only thing left was to determine how these two molecules interacted together.
Porter immunized goats with either the Fab or the Fc portions of antibody. In this way the goats made antibodies in
their serum against either Fab or Fc. When the serum from the goats was reacted against antibody, Porter found
that goats immunized with Fab had antibody against heavy chain and light chain but goats immunized with Fc only
had antibody against heavy chain.
These series of experiments helped to determine the structure of antibody as we know it today.

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GENERAL FUNCTIONS OF IMMUNOGLOBULINS
A. Ag binding - Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin
actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of
antibodies and can result in protection of the host.
Valency - The valency of antibody refers to the number of antigenic determinants that an individual antibody
molecule can bind. The valency of all antibodies is at least two and in some instances more.
B. Effector Functions - Often the binding of an antibody to an antigen has no direct biological effect. Rather, the
significant biological effects are a consequence of secondary "effector
functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the ability to
carry out a particular effector function requires that the antibody bind to
its antigen. Not every immunoglobulin will mediate all effector functions.
1. Fixation of complement - lysis of cells, release of biologically active molecules
2. Binding to various cell types - phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors
that bind immunoglobulins and the binding can activate the cells to perform some function. Some
immunoglobulins also bind to receptors on placental trophoblasts. The binding results in transfer of the
immunoglobulin across the placenta and the transferred maternal antibodies provide immunity to the fetus and
newborn

Different antibody isotypes or classes:

IgG:
Structure - The structures of the IgG subclasses are presented in Figure . All IgG's are monomers (7S
immunoglobulin). The subclasses differ in the number of disulfide bonds and length of the hinge region. In
humans the four different subclasses of IgG can be distinguished by the size of their hinge region and the position
and number of disulfide bonds. They can also be distinguished by function.

IgG1, 3 and 4 cross the placenta and protect the developing

fetus. IgG3 is the most effective at activating complement.
IgG4 is not able to activate complement at all. IgG1 and
IgG3 bind Fc receptors on phagocytic cells thus mediating
opsonization. IgG4 and IgG2 are not efficient opsonins.
Properties - Most versatile immunoglobulin because it is capable of carrying out all of the
functions of IgG molecules.
a) IgG is the major Ig in serum - 75% of serum Ig is IgG
b) IgG is the major Ig in extra vascular spaces
c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by receptor on
placental cells for the Fc region of IgG. Not all subclasses cross equally; IgG2 does not cross well.
d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement

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e) Binding to cells - Macrophages, monocytes, PMN's and some lymphocytes have Fc receptors for the Fc region of
IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to
the Fc receptors on PMN's, monocytes and macrophages is that the cell can now internalize the antigen better. The
antibody has prepared the antigen for eating by the phagocytic cells. The term opsonin is used to describe
substances that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of
cells results in the activation of other functions.

IgM:
Structure - The structure of IgM is presented in Figure .
IgM normally exists as a pentamer (19S immunoglobulin)
but it can also exist as a monomer. In the pentameric
form all heavy chains are identical and all light chains are
identical. Thus, the valence is theoretically 10. IgM has an
extra domain on the chain (CH4) and it has another
protein covalently bound via aS-S bond called the J chain.
This chain functions in polymerization of the molecule
into a pentamer.
Properties
a) IgM is the 3rd most common serum Ig.
b) IgM is the first Ig to be made by the fetus and the first
Ig to be made by a virgin B cells when it is stimulated by
antigen.
c) As a consequence of its pentameric structure, IgM is a
good complement fixing Ig. Thus, IgM antibodies are
very efficient in leading to the lysis of microorganisms.
d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in
clumping microorganisms for eventual elimination from the body.
e) IgM binds to some cells via Fc receptors.
f) B cell surface Ig - Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-
terminal end to anchor it into the membrane (Figure ). Cell surface IgM functions as a receptor for antigen on B
cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B cell called Ig-
and Ig- as indicated in Figure . These additional proteins act as signal transducing molecules since the cytoplasmic
tail of the Ig molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an
antigen is required before a signal can be transduced by the Ig- and Ig- chains. In the case of T-independent
antigens, contact between the antigen and surface immunoglobulin is sufficient to activate B cells to differentiate
into antibody secreting plasma cells. However, for T-dependent antigens, a second signal provided by helper T cells
is required before B cells are activated.
IgM makes up 5-10% of all serum antibody. IgM is monomeric if membrane bound. Secreted IgM is a pentamer,
made up of 5 monomeric IgM molecules. Each pentameric IgM contains an additional Fc-linked polypeptide called
the J chain (joining chain). This is necessary for pentameric IgM to form and is added just before secretion.
IgM is the first immunoglobulin class to be made during a primary response. It is the first immunoglobulin made by
a neonate. Because IgM is pentameric, it has 10 antigen binding sites thus can bind 5 molecules of antigens. Very
efficient binding viral particles.

IgA:
1. Structure - Serum IgA is a monomer but IgA found
in secretions is a dimer as presented in Figure. When IgA
exits as a dimer, a J chain is associated with it.

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When IgA is found in secretions is also has another protein associated with it called the secretory piece or T piece;
sIgA is sometimes referred to as 11S immunoglobulin. Unlike
the remainder of the IgA which is made in the plasma cell, the secretory piece is made in epithelial cells and is
added to the IgA as it passes into the secretions. The secretory piece helps IgA to be transported across mucosa and
also protects it from
degradation in the secretions.
Properties
a) IgA is the 2nd most common serum Ig.
b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is
found in secretions secretory IgA is important in local (mucosal) immunity.
c) Normally IgA does not fix complement, unless aggregated.
d) IgA can binding to some cells - PMN's and some lymphocytes.
Makes up 10-15% of serum antibody but is the major antibody found in external secretions such as breast milk,
saliva, tears and mucus. Predominately found as a dimer.

The IgA of external secretions is called secretory IgA (sIgA). Like

IgM sIgA has a J chain that facilitates dimerization. It also has a
polypeptide chain called secretory component. Secretory
component is derived from the receptor that is responsible for
IgA's transport across cell membranes. This receptor is called the
poly-Ig receptor or (pIg-R). The daily production of sIgA is greater
than any of the other immunoglobulins. The plasma cells that
make sIgA preferentially migrate to subepthelial tissue such as the
gastrointestinal tract, the urogenital tract and the respiratory tract.
When sIgA is made, it is bound by pIg-R expressed on the surface
of epithelial cells. It is then transported across the cell membrane
in a vesicle. When it reaches the external surface or lumen,
enzymatic cleavage releases sIgA along with secretory component.
This process is called receptor-mediated endocytosis. Secretory
component is important because it masks sites in the IgA hinge
region that are susceptible to enzymatic cleavage making the IgA
molecule more resistant in an environment that is full of proteases,
such as the gastrointestinal tract. The pIg-R binds the J-chain if
sIgA and IgM to facilitate cellular transport of these two antibodies. IgA is often one of the first lines of defense
against pathogens that enter through mucosal surfaces. Because it is dimeric, it more efficiently binds viral and
bacterial antigens preventing them from binding on the surface of mucosal cells and inhibiting infection. IgA is also
abundant in breast milk. This is important early in a baby's life when he or she does not yet have a fully functioning
immune system.

IgE:
Structure - The structure of IgE is presented in Figure . IgE exists as a monomer and has an extra domain in the
constant region.
Properties
a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even
before interacting with antigen. b) Involved in allergic reactions - As a consequence of its binding to basophils an
mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release
of various pharmacological mediators that result in allergic symptoms.

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c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE
levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils
to IgE-coated helminths results in killing of the parasite.
d) IgE does not fix complement.
IgE antibodies mediate immediate hypersensitivity reactions and are responsible for the symptoms of hay fever,
asthma, hives, and anaphlylactic shock.
IgE binds the Fc receptors of basophils and mast cells. These Fc receptor bound IgE molecules are then
crosslinked by an antigen or allergen. This induces degranulation of the basophil and/or mast cell and various
pharmocological factors associated with allergies (histamine for example) are released.

IgD:
Constitutes only 0.2% of serum antibody. Is a major membrane-bound form of antibody on B cells. May function
in B cell activation. No major immune effector function for IgD has yet been found. Structure - The structure of
IgD is presented in the Figure. IgD exists only as a monomer.
Properties
a) IgD is found in low levels in serum; its role in serum uncertain.
b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B
cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig- and Ig-
chains.c) IgD does not bind complement.

Q. What do you mean by isotypic , idiotypic and allotypic variations of antibodies ? Or,
antibodies can be regarded as antigens-
justify the statement.
Antigenic Determinants on Immunoglobulins
Because antibodies are glycoproteins, they can be
immunogens. Anti-immunoglobulin antibodies have
been powerful tools in studying humoral immunity.
Antibody antigenic determinants fall into three
categories:

isotype- constant region determinants that define the

antibody class.
Isotypes differ from species to species. Therefore,
when antibodies from on species is injected into
another species the recipient sees the isotypic
determinants as foreign. Anti-isotype antibody can be
used to determine class of antibodies found in serum.

allotype- Although within a species the isotype genes

are the same the genes have multiple alleles that differ
by subtle amino acid differences. These are called
allotype determinants. Antibodies against allotypic
determinants can be generated by injecting
immunoglobulin from one individual of the same
species into a different individual of the same species.
They are also found on the constant regions.

Idiotype- this is the unique amino acid sequence

found in the variable regions of the H and L chains at
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the antigen-binding site. These are called idiotopes. They may be in the antigen-binding site or outside the antigen-
binding site. They are conformational. Each antibody will have several idiotopes. The sum of the idiotopes is the
idiotype.
What are B-cell receptors? Add a note on monoclonal antibodies and its applications.
B cell receptor (BCR):
The BCR is a multi-protein transmembrane complex that includes surface antibody. The other proteins are a
disulfide-linked heterodimer called Ig-alpha/Ig-beta.
Ig-alpha and Ig-beta have long cytoplasmic tails that interact with the intracellular signalling machinery. The
membrane Ig molecule does not have a cytoplasmic tail long enough for cell signalling and needs the Ig-alpha/Ig-
beta heterodimer. There are several membrane bound proteins that share a similar structure to surface antibody.
Therefore, they have been grouped in the immunoglobulin superfamily.

Monoclonal antibodies:
Because antigens possess many B cell epitopes, the immune response generated produces many different types of
antibodies. This is known as a polyclonal response because there are many different antibodies derived from
different B cells recognizing different epitopes. For research and therapeutic purposes it is sometimes necessary to
use monoclonal antibodies. These are antibodies that recognize only on epitope of an antigen and are derived from
a single B cell clone.
Clinical uses of monoclonal antibodies include:
Detection of tumors- breast cancer specific monoclonal antibodies can be labeled with a tracer, injected into the
body and then located. This may help in determining whether cancer has spread.
Immunotoxins- these are antibodies against tumor antigens that have been coupled to toxins such as ricin, Shigella
toxin and diptheria toxin. These inhibit protein synthesis and kill cells. In this manner the antibody acts to deliver
the toxin to the specific cell which then dies due to an inability to synthesize the necessary proteins.

Q. Compare briefly about the different classes of immunoglobulins. What are the
physiological functions of immunoglobulins?
Some antibody classes form multimeric structures, pentamers in the case of IgM and dimers or trimers in the case
of IgA. These two isotypes also associate with a small protein called the joining (J) chain required for stabilisation of
the complexes.
Antibody functions:
Opsonization
Opsonization refers to the coating of antigen with antibody that results in enhanced uptake of the antigen. There
are two receptors capable of binding opsonized antigen. One is the
FcR, see above, the other is the receptor for one of the complement
components C3b. If an antigen is coated with IgM antibody which
has C3b bound to it then it will be opsonized via the C3b receptor.
Ligation of the FcgammaR on phagocytes, during the process of
phagocytosis of opsonized particles, leads to their activation and the
enhanced uptake and degradation of antigen.
Antibody dependent cellular cytotoxicity (ADCC)
Neutrophils, eosinophils, phagocytes and NK cells all mediate
ADCC. Ligation of the low affinity FcgammaRIII (CD16) molecule
activates the lytic machinery. ADCC is only triggered by complexes
of antigen and antibody. In this way, Fc regions form an array
sufficient to increase the avidity of the interaction thus avoiding
ADCC being triggered by free immunoglobulin.
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Helminths cannot be killed by ADCC. Eosinophils, however, produce a basic protein which kills helminths and
these cells express an Fc receptor for IgE. IgE provides some protection against helminths.
Immediate (type I) hypersensitivity
Caused by IgE antibodies. Ligation of the Fc epsilonR1 receptor on mast cells and basophils leads to the
production of inflammatory nd vasoactive mediators (histamine), lipid derived mediators (leukotrienes,
prostaglandins, platelet activating factor etc.) and cytokines. This phenomenon manifests as allergy but is clearly of
benefit in clearance of extracellular parasites.
Secretory Ig
Epithelial cells in the intestine and other secretory epitheilia (tear duct, salivary gland, lactating mammary gland etc)
possess a receptor for polymeric Ig, which mediates secretion of antibody, predominantly IgA also IgM into the
external secretions. Secreted IgA forms a very important defence against infection.

Neonatal immunity
In Humans, maternal IgG can cross the placenta. Babies have a high level of maternal IgG at birth. They also
acquire IgA (and some IgG) postnatally from breast milk, however these remain in the gut lumen.

In some other species for example ruminants and rodents, IgG does not cross the placenta. Instead high levels of
IgG are secreted in the milk produced perinatally (colostrum) and IgG is specifically taken up from the gut into
blood of the nenonate via an IgG receptor, whereas IgA remains in the gut.

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In all species maternal IgG,IgA are important both locally and systemically in protecting the neonate during the
development of it's own immune system.

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