 Structure
of Immunoglobulins (cont)
Hinge region and interchain
disulfide bonds Click the image below for a 3-D tutorial on Ab structure. Requires Chime.
Prolines may keep the two combining sites of the molecule separated in space and contribute to the molecular mobility of the various parts of the Ig.
Intra chain disulfide bonds and structural domains inter - between chains Domains : conserved units of molecular structure witin a protein that confer a unique motif. There are regularly spaced
cysteines in the amino acid sequence of both Heavy and Light chaines
of immunoglobulin which allow for the formation of regularly spaced
intrachain disulfide bonds. The looped segments which are created
give a unique shape to Ig molecule, resulting in structural domains.
We have been talking about the Ig domains in structural terms but they have functional relevance as we will see. 3D structure : X Ray crystallographic
analysis has revealed the overall 3D picture of the molecule. All Igs are composed of the
four-chain structure (H2L2) described so far.
Structural Basis of Antibody Activity It was possible to deduce Immunoglobulin chain structure and the organization of the polypeptides into structural domains using the various biochemical approaches described earlier. However, uinderstanding this, immunologists still didn't have any knowledge of how the Ig actually binds to antigen. One of the major problems facing immunologists was the heterogeneity of immunoglobulins present in serum. In order to determine amino acid sequence and to understand how Igs were able to show specificity for antigen, it was necessary to obtain a homogeneous preparation of Ig. A preparation in which all Ig molecules present were identical in amino acid sequence. Multiple Myeloma Not only do Multiple Myeloma patients have massive quantities of 1 type of immunoglobulin in their serum (so-called myeloma proteins) but they also excrete intact light chains in their urine. These secreted light chains are termed Bence Jones proteins. Investigators used the Bence Jones proteins to perform the very first amino acid sequencing experiments. Importantly, they then compared the amino acid sequences of Bence Jones proteins between different patients. Sequencing results:
amino terminal half of the light chain- variable in sequence The amino terminal half of
the light chain was termed the variable domain (VL)
Susequent amino acid sequence
analysis of heavy chains showed that they too were divided into constant
and variable regions. These findings led to an
understanding that there is a structural basis for the diversity of
the antibody molecule. Within VL and VH there are "hot spots" of variability. Variability
=
# of different amino acids at a given position These hot spots of variabilty
were termed hypervariable regions As you might have guessed by now, these regions form the antigen binding site of the immunoglobulin molecule. Remember: So, antigen-binding ability resides in the portions of the antibody molecule which are hypervariable. 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 maintained. Antibodies in contrast, must be able to bind with an enormous number of antigens, so the active site varies to allow this to happen. Light Chains k and l Mice have 95% k and 5% l Humans have 60% k and 40% l In contrast, there are several different types of heavy chains: Heavy Chains 5 Different Classes (Isotypes)
IgG g Each differs in the heavy
chain which is used. Variation of molecular weight is due to different #s of monomers and due to different levels of glycosylation. Common property: Each class has ability to combine with antigen. IgG
Represents 70% of total
serum Ig - major class of Ig in serum of normal adults. In Mice, subclasses are designated:
IgG is only class of Ig which
is able to cross the placenta in humans and other primates For this reason, human IgG
is termed Maternal Antibody = class of maternal antibodies which
protects the newborn.
IgM exists as a monomer in the membrane-bound form on the surface of B lymphocytes. All mature, but naive B lymphocytes
possess both IgM and IgD in the membrane-bound form. IgM exists as a Pentamer with a ~mw 850,000 to 1 million (each monomer ~180,000). Monomers are joined together
through disulfide bridges.
J chain ~15,000 (serves like
a clasp) IgM represents ~10% of total
serum Ig Rich in carbohydrates, constitutes
12% of its weight IgA
Serum IgA
Secretory
IgA The secretory component (or
piece) is synthesized by mucosal epithelial cells and is derived from
the poly-Ig-receptor. Due to the huge surface area of the body covered by mucosa, the combined quantities of serum and secretory IgA make this the most abundant in the human body. IgA also is the predominant
class of Ig found in seromucous secretions, saliva, tracheobronchial
secretions, colostrum, milk, and genitourinary secretions. IgD
IgE Monomer mw~180,000
IgE also binds to Fc receptors on the surface of eosinophils. This allows eosinophils to participate in ADCC (antibody dependent cell-mediated cytotoxicity) reactions against parasitic helminths. Like IgM, IgE has 4 CH domains
in the e chain ANTIGENIC DETERMINANTS ON IMMUNOGLOBULINS Because immunoglobulins are large glycoproteins, they can function as excellent immunogens. 3 categories of epitopes (or antigenic determinants) have been described for immunoglobulins: Isotypic Determinants
examples: Goat anti-
mouse IgG (antibody raised in a goat specific for isotypic determinants
associated with the gamma heavy chain of mice) Allotypic Determinants
Allotypic determinants are due to subtle amino acid differences encoded by different alleles for Ig. Antibody to allotypic determinants can be produced by injecting antibodies from one member of the same species into a different member of the species who possesses a different allotype of Ig. [May be produced by mothers during pregnancy in response to paternal allotypic determinants on fetal Ig] [May also be produced following blood transfusion if Igs are not completely removed from donor blood] Idiotypic Determinant
Each determinant is referred
to as an idiotope Sum of all idiotopes = idiotype To get an anti-idiotypic antibody you must minimize isotypic and allotypic differences by using a Syngeneic Recipient or even same animal. Substantial evidence suggests that the immune response uses anti-idiotypic antibodies to regulate the humoral response.
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IgM
