Important concept: For any given antibody molecule, the two Heavy chains will be identical to each other and the two Light chains will be identical to each other.
Hinge region and interchain disulfide bonds
The region between the papain and pepsin cleavages is called the hinge region. This region rich is in prolines and cysteines, which serves several functions:
cysteines contribute to interchain disulfide bridges
*Prolines keep the two combining sites of the molecule separated in space and contribute to the molecular mobility of the various parts of the Ig. The Fab arms of IgG in particular are able to "wag" and "wave" which allows them to come into contact with antigen.
All Immunoglobulins are composed of the basic four-chain structure described previously. However, some classes of Ig are composed of polymers of the H2L2 basic monomeric form.
Intra chain disulfide bonds and structural domains
inter - between chains
intra - within a polypeptide chain
There are regularly spaced cysteines that result in intrachain disulfide bonds
they give unique shape to Ig molecule, resulting in structural domains. Each looped domain contains approximately 60 amino acids.
Domains : conserved units of molecular structure witin a protein that confer a unique motif. Any protein which possesses the domain structure of immunoglobulins is classified as a member of the Ig superfamily of proteins. Proteins in this family have at least one of the looped domains stabilized by intrachain disulfide bonds, characteristic of immunoglobulins. Some examples of such proteins are the Class I and Class II MHC proteins, the T cell receptor, and a variety of the CD proteins found on lymphocytes.
Back to myeloma patients. Not only do they have massive quantities of 1 type of immunoglobulin in serum but they also excrete light chains in their urine. Bence Jones proteins. Investigators used the Bence Jones proteins to perform the first amino acid sequences. Importantly, they then compared the amino acid sequences between patients.
Results: amino terminal half of the light chain- very variable
carboxyl terminal half of the chain - very constant
Each light chain domain consists of approximately 100-110 amino acids. The amino terminal domain is termed the Variable light domain (VL) whereas the carboxyl terminal half of the light chain is termed the Constant light domain (CH).
It was also determined that there were actually two different classes of light chain constant regions. These two types of light chains have been designated Kappa and Lambda. In humans, approximately 60% of the immunoglobulins contain kappa light chains while 40% contain lambda light chains. In mice, ~95% of Igs use the kappa light chain and only 5% use the lambda light chain.
Analysis of heavy chains showed that they too were divided into constant and variable regions. The variable region, again, was restricted to a region of ~100-110 amino acids in the amino terminal end of the heavy chain. The remaining amino acid sequence was much more constant in amino acid sequence. There are either 3 or 4 constant heavy region domains (designated CH) depending on the immunoglobulin isotype. Five different classes (isotypes) of the heavy chain constant regions were identified and were designated as mu, gamma, delta, epsilon, and alpha. Mu and epsilon heavy chains each have four constant heavy region domains (CH1, CH2, CH3 & CH4) while gamma, alpha, and epsilon each have only 3 constant heavy region domains.
These findings led to an understanding that there is a structural basis for the diversity of the antibody molecule. If each antibody molecule can differ from every other antibody molecule at a large number of amino acid positions, we can see how the basic 4-chain structure can give rise to an astronomical number of different functional molecules. The variation of a few amino acids in the V region of an Ig must be responsible for antibody diversity and specificity.
Within VL and VH there are "hot spots" of variability.
Variability = # of different amino acids at a given position /
frequency of the most common amino acids at that position
These hot spots of variabilty were termed hypervariable regions. The hypervariable regions represent ~15-20% of the V region sequence. The remaining sequences with very little variability were termed framework regions which represent the remaining 80-85% of the sequence.
The hypervariable regions of the heavy and light chains together form the antigen binding site of the immunoglobulin molecule. For this reason, the hypervariable sequences are also termed CDRs (complementarity determining regions)
So, antigen-binding ability resides in the portions of the antibody molecule which are variable. With enzymes, in contrast, the active site is the least variable region of the molecule. All cytochrome C molecules in all species must carry out only one function, and the sequence required for that function is highly conserved. Antibodies in contrast, must be able to bind with an enormous number of antigens, so the active site varies to allow this to happen.
In humans there are 5 different Classes (Isotypes) which are determined by the properties of the constant region domains of heavy chains.
IgG gamma
IgM mu
IgD delta
IgA alpha
IgE epsilon
Each isotype differs in the heavy chain which is used. Chains are designated by a small greek letter. Each chain is coded by its own constant region gene segments.
Common property: Each isotype has ability to combine with a specific epitope.
IgG
exists only as a monomer with a molecular mass of ~ 150,000 D.
It is present in membrane-bound form on the surface of B lymphocytes and in secreted form in the serum.
70% of total serum Ig - major class of Ig in serum of normal humans.
IgG is a good agglutinating antibody (largely due to the presence of the hinge region which allows for Fab arm movement.
In addition, the IgG isotype is able to fix complement when bound to particulate antigen.
In humans, there are 4 subclasses:
IgG1
IgG2
IgG3
IgG4
In the Mouse, the four subclasses are:
IgG1
IgG2a
IgG2b
IgG3
IgG is only class of Ig which is able to cross the placenta in humans and other primates. Many mammals do NOT have placental transfer of maternal antibody.
Maternal Antibody = class of maternal antibodies which protects the newborn.
One potential drawback -----> RH disease
IgM
exists as a monomer on the surface of the B lymphocyte and as a pentamer when secreted by plasma cells.
pentamer has a molecular mass of ~ 850,000 to 1 million D while each monomer is ~180,000D.
IgM represents ~10% of total serum Ig and is always the first isotype of antibody synthesized during a primary humoral response.
Carbohydrates constitute~ 12% of the weight of the IgM protein.
The mu heavy chain contains 4 CH domains.
Due to the presence of 10 identical antigen binding sites, IgM is an excellent agglutinating antibody. In addition, IgM is very efficient at fixing complement.
The individual monomers are joined together through interchain disulfide bridges. In addition, a small polypeptide known as J chain ~15,000D serves like a clasp in holding the pentamer together. The1st monomer and the last monomer are attached to the J chain.
See figure in text book
Serum IgA
IgA may exist as a monomer or dimer in serum.
80% of the proteins in serum are in the monomer form, while ~20% are in the dimer form.
monomer has a molecular mass of ~ 160,000 D
In the dimer form, the two monomers are held together by a J chain.
The dimer has a molecular mass of ~ 415,000D
Secretory IgA
IgA which is present in the mucous secretions of the G.I. tract, respiratory tract, and the urogenital tracts.
Due to the large surface area represented by mucosal epithelium, IgA is the most abundant isotype of Ig in the body.
There are two isotypes ( IgA1 and IgA2 ) in humans.
The secretory IgA possesses a J chain as well as a 70,000 D secretory component or secretory piece.
The secretory component is synthesized by mucosal epithelial cells and is covalently linked to the Fc of the IgA - J chain dimer as it passes through the epithelial cells of the mucosa into the lumen. The presence of the secretory component renders the IgA much less susceptible to proteolysis in the hostile mucosal environment.
IgA is the predominant Ig in seromucous secretions, saliva, tracheobronchial secretions, colostrum, milk, and genitourinary secretions.
IgA in colostrum and breast milk does not enter the baby's circulation but protects the infant's GI tract and perhaps respiratory tract from pathogens.
IgD
IgD exists almost exclusively as an antigen receptor on the surface of B lymphocytes. In the secreted form it is extremely labile - much more sensitive to proteolysis than the other Ig classes.
IgD exists only as a Monomer with a molecular mass of ~180,000D.
IgD is found to be present in association with IgM on the surface membrane of B lymphocytes. A cell that expresses both IgM and IgD is a mature but "virgin" B cell. The IgM and IgD molecules on the same cell have the same antigen-binding specificity. In other words, the IgD and IgM on the surface of a single B lymphocyte have the same VL and VH region sequences.
IgE
IgE exists only as amonomer with a molecular mass of ~180,000D.
IgE is termed Reaginic or (homocytotropic) antibody due to its role in Type 1- immediate type hypersensitivity ( allergy).
It is present in extremely low concentrations in serum.
IgE has a profound effector function due to the fact that basophils and mast cells have high affinity receptors for the Fc region.
Consequences: Allergies due to mast cell degranulation and release of pharmacologically active substances.
Like IgM, IgE has 4 CH domains.
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