Antibodies Flashcards
Define: H chain L chain kappa and lambda chains hinge region Fab, F(ab')2, Fc complementarity-determining regions hypervariable regions variable (V) and constant (C) domains VL and CL VH and CH Name the 5 antibody classes and their characteristic heavy chains Distinguish the immunoglobulin classes IgG, IgA, and IgM in terms of size and approximate concentration in serum. Describe the organization of the immunoglobulin genes in the chromosome. Describe the mechanisms by which the
IgA
dimer of 2h and 2 L, held together by a J chain
In any one antibody, the H chains are identical, and so are the L chains, so the molecule is (rotationally) symmetrical.
IgM
pentamer of 2h and 2 L, held together by a J chain
In any one antibody, the H chains are identical, and so are the L chains, so the molecule is (rotationally) symmetrical.
H chains
The 5 kinds of H chains (gamma, alpha, mu, epsilon, delta) define the class of antibody to which the molecule belongs, and therefore its biological properties
L chains
come in 2 varieties: kappa or lambda (the original light chain gene family has duplicated long ago)
Although each cell that makes an antibody has a choice of using kappa or lambda, it uses only one kind. So, for example, an IgA molecule will be kappa or lambda type, while another IgA might be the other.
What about a cell switching the antibody its making?
A single cell may switch from making IgM to making IgA, for example. When this
happens, the heavy chain changes (mu replaced by alpha) but the L chain, either kappa or
lambda, stays the same during the switch.
constant region
When the amino acids in many antibody molecules are sequenced, you see that for each chain type there is a region that is essentially identical, no matter what the specificities of the antibodies were. This is the constant region, and it is made up of 1 (in L
chains) to 4 (in epsilon and mu) compact, structurally-similar domains called C domains.
variable domain
Each chain also has, at its N-terminal, a domain that is different in sequence between antibodies of different specificities: the variable domain or V. The antibody’s combining site, which binds antigen, is made up of the V domains of both the H and L chain (VH and VL.)
hypervariable and CDR, complementarity-determining regions
Amino acid sequence variability is not distributed uniformly along the V domain; most of the variability is in 3 areas called, therefore, hypervariable regions. It is more functionally
significant to call them CDR, complementarity-determining regions, because the amino acids in the hypervariable regions comprise the actual antigen-binding site.
Valence
Valence refers to the number of antigenic determinants (epitopes) an antibody molecule can theoretically bind. What is the valence of IgG? of secreted IgA? of IgM? Of Fab? of F(ab’)2? How about of an isolated VL or VH?
allotypes
There are minor allelic differences in the sequence of immunoglobulins between individuals, just as blood types or eye color differ.
These differences are called allotypes, and the allotypes you express are determined by the allotypes your parents had, in the usual Mendelian fashion.
Allotypes are useful in genetics, for example in determining relatedness, and sometimes in forensic medicine. Occasionally, an immunodeficient patient getting immunoglobulin treatments will make antibodies to someone else’s allotype; this could be awkward. If certain allotypes function more efficiently than others, it could explain why some
people are more susceptible to some infections than other people; we don’t know much about that yet, though there is some evidence for the idea.
Idiotypes
Each antibody will have its unique combining region, made up of the CDR amino acids of its L and H chains; we can call this unique structure an idiotype (idio means self). It might not surprise you that, under rather special circumstances, antibodies can be made (most
easily in another species) that recognize the unique sequence of that combining site, and no other. Such an antibody is an anti-idiotype. In other words, it is almost completely correct to say that an idiotype is an antibody’s unique combining site considered as an antigen.
IgG
the main antibody in blood and tissue fluids. It neutralizes toxins and blood-borne viruses, binds bacteria and facilitates their destruction by activating complement and by binding them to phagocytic cells.
good at: complement fixation, bacterial lysis, and antiviral activity
great at: toxin neutralization
IgA
can do similar things in the blood (as IgG), but its real role is as the dimer form in secretions, where secretory component protects it from proteolysis.
good at: bacterial lysis
great at: antiviral activity, toxin neutralization
IgM
does much the same as IgG. It is the first antibody to appear in the serum after
immunization, and it is very efficient at activating complement. It does not get into tissue fluids very efficiently, nor is it bound efficiently by phagocytic cells.
good at: antiviral activity
great at: complement fixation, bacterial lysis
IgD
IgD’s role in blood, if any, is uncertain; it seems to function mainly as a receptor on naïve B
cells.
IgE
IgE is the antibody which causes Type I immunopathology, also called immediate
hypersensitivity or allergy. Its true importance is in resistance to worms and other parasites
great at: mast cell/basophil degranulation
Antigen-Antibody Interaction
When an IgG or IgM antibody binds antigen with at least one of its binding sites, there may be a change in the angle between the two Fab parts, so that the molecule may be more Y or T shaped than before (this explains why the region between Fabs and Fc is called the Hinge.) This results in a bulging of the structure of the Fc part so that one or two very important biological activities are initiated:
- Binding to phagocytic cells, especially PMNs, eosinophils, and macrophages, which have
receptors (FcR) for the altered Fc of IgG (but not of IgM), and - C1q, the first component of the complement system, now binds to two adjacent Fcs and is activated. Note: IgGs will have to be binding close together on the same (usually
bacterial) surface, but one IgM can do it alone, because it carries 5 Fcs at all times. This makes IgM much better at activating complement.
It is always helpful to think about antigen-antibody interactions in steps: first there is binding (recognition), and then the antibody can do something else, like cross-link two antigen molecules, or activate complement, or bind to a phagocyte (function). Some important defense mechanisms, and also useful tests, depend only on the first step, and others involve the secondary events as well.
Biological Functions of IgM
Good at virus neutralization
Poor at toxin neutralization
Excellent at bactericidal activity
Excellent at causing agglutination of antigens
Excellent at causing precipitation of antigens
Excellent at complement fixation
Does not bind to macrophage Fc receptors
As a monomer it can serve as a surface receptor for antigens on B cells (like IgD)
Elevated levels indicate a recent infection or other exposure to antigen
Does not cross the placenta
Not useful for protecting immunocompromised individuals
Not present in interstitial fluids (too big)
Can be present in bodily secretions
Biological Functions of IgG
Good at virus neutralization
Excellent at toxin neutralization
Good at bactericidal activity
Good at causing agglutination of antigens
Good at causing precipitation of antigens
Good at complement fixation
Binds to macrophage Fc receptors
Crosses placenta providing protection to the fetus.
Can mediate hemolytic disease of the newborn (blue baby syndrome, Rh mismatch)
Can be used for protecting immunocompromised individuals (gamma globulin)
Can be used as a blocking antibody to block TNF production (rheumatoid arthritis)
Can be used as a blocking antibody to block allergens (desensitization to hypersensitivity)
Not present in most bodily secretions
Present in interstitial fluids
Biological Functions of IgA
Excellent at virus neutralization
Excellent at toxin neutralization
Good at bactericidal activity
Good at causing agglutination of antigens
Good at causing precipitation of antigens
Does not bind to macrophage Fc receptors
Daily production of IgA is greater than any other Ig
B cells that produce IgA migrate to the subepithelial tissue of most mucosal epithelia and of glandular epithelia
Present in bodily secretions
Present at very high levels in colostrum and present in breast milk. Provides an excellent level of protection of newborns against respiratory and intestinal infections
Biological Functions of IgE
Cross-linking of IgE molecules on the surface of a mast cell or basophil causes the release of histamine; the synthesis of prostaglandins, leukotrienes, and other chemokines and cytokines
IgE plays a major role in combating parasitic infections
IgE plays a role in combating pulmonary fungal infections
Individuals who express allergies to certain antigens over-produce IgE to those antigens.
This causes a high level of expression of IgE with the same paratopes (recognize the same epitope on an antigen) on given mast cells. This makes it easier to cross-link two IgE molecules.
When antigen is present, many mast cells are degranulated, resulting in an over-stimulation of the immune system that is manifested as an allergic reaction (Type 1 hypersensitivity)
IgE also plays a role in asthma.
Keys to Antibody Diversity
Antibody diversity is generated during genetic rearrangement by mixing and matching one of each of the various gene segments for the heavy and light chains in a combinatorial manner.
Antibody diversity is generated by variation incorporated at the joining sites for the various segments of the heavy and light chains.
Antibody diversity is generated by hypermutation in one of the gene segments (variable regions) of the heavy and light chains during proliferation of B cells.
Antibody diversity is generated by mixing and matching heavy and light chains in a combinatorial manner.
Recombination
The variable domain region of heavy chain genes is broken up into multiple V, D, and J gene segments; the V region of light chains into V and J segments; so generically we say ‘V(D)J.’ The cell will choose one of its Vs, one D, and one J to make a VH domain gene.
How to make Heavy Chains
The developing B cell first brings one random D segment close to one J; the DNA is cut, the intervening DNA is discarded and the ends joined. It then brings a V segment up to the recombined DJ, and repeats the cutting and joining process (there are splice acceptor and donor sites adjacent to each segment). Then the entire region from the assembled
VDJ unit through to the end of the delta (of IgD) constant region gene can be transcribed into RNA. These primary RNA transcripts are alternatively processed using alternative polyA sites and splicing, first to make only VDJ-μ, and later to make both VDJ-μ and VDJ-δ messages.
How to make Light Chains
Gene rearrangement is similar, but they have only V and J segments, no D; and only one C domain gene.
RAG recombinases
The enzymes that do the recombination of antibody and T cell receptor DNA are called RAG-1 and RAG-2 recombinases. The recombinases first bind splice signals to the right of a D segment and the left of a J segment, pull them together, and then cut and splice. Then they look for a splice sequence to the right of a V segment and do it again. If RAGs are knocked out, mice make neither B nor T cells. It happens in humans, too—very rarely (Omenn Syndrome).
Joining area variation
The production of the V-D and D-J joints are ‘sloppy.’ The cell has randomizing mechanisms:
First, exonucleases can chew away a few nucleotides after the DNA is cut but before two gene segments (D to J, V to DJ) are joined.
Second, the cell can add a few nucleotides as well with an enzyme called terminal deoxynucleotidyl transferase (TdT) which doesn’t use a template so its additions are random.
Thus you can’t predict the sequence at the joining area (which is called an ‘N’ region).
Let’s say that V7 has joined to D2 but in one cell there’s an extra alanine and tyrosine there and in another one there is a leucine missing. This produces a lot more completely random diversity.
There is a price for it: two times out of three the N region, being of random length, will create a frame-shift mutation, that is, a nonsense codon which terminates transcription.
Receptor editing
Although a B cell tries to rearrange each allele just once, when a rearrangement is detected as faulty (say a stop codon is generated), or when an anti-self receptor has been displayed, if the recombinases (RAG genes) are still active it can ‘try again.’ Sometimes this results in a successful cell. The process is called receptor editing
Ig Molecules Expressed on Mature B Cells
Mature (but not activated) B cells initially express IgD and IgM on their external surfaces (these are B cell receptors). The choice of IgD and IgM occurs at the level of processing of mRNA, so a given B cell can both express IgD and IgM. As mature B cells are activated to divide and differentiate by their cognate antigen, they switch from membrane-bound IgD and IgM to secretory IgM. This switch occurs at the level of processing of mRNA transcripts. As they continue to divide and differentiate, they may undergo additional class switching from IgM to IgG, or IgE, or IgA. These last switches occur at the level of rearrangements of DNA.
affinity
maturation
Selection of the best-fitting mutants after antigenic stimulation allows a gradual increase of affinity during an immune response—an exceptionally nice design feature called affinity
maturation. (For T cells, there’s no somatic mutation after contact with antigen.)
How it works:
Activation-Induced (Cytidine) Deaminase (AID) converts random cytosines in the CDR gene regions to uracil. So a C:G pair becomes a uracil: guanine mismatch. The uracil bases are excised by the repair enzyme uracil-DNA glycosylase. Error-prone DNA polymerases then fill
in the gap, creating mostly single-base substitution mutations, so at the end of cell division one daughter may be making a different (worse? better?) antibody.
Class Switching
After activation, B cells switch from membrane-bound IgM and IgD to secreted IgM by differential splicing.
As the activated B cells continue to divide, they class switch to production of IgG by DNA rearrangement (again).
Activated B cells may continue to class switch to production of IgE or IgA by further DNA rearrangement.
A single mature B cell starts by making both IgM and IgD, which it puts into its membrane as receptors, and then later it may switch to making IgG, IgE, or IgA. In all cases, the L chain and the VH domain stays the same but the C region of the H chain changes.
What happens is that the cell which has put its particular H-chain VDJ combination together with its mu and delta genes goes back to its DNA, does a loop-out of mu and delta, and puts VDJ next to the C region gene of gamma or epsilon or alpha, while excising and discarding intervening DNA. The new mRNA, then, may be VDJα or VDJγ or VDJε. Thus a cell which is making IgM can go on to make IgG, but a cell making IgG cannot go back to making IgM; the mu information is physically gone. ‘M to G’ or ‘M to A’ or ‘M to E’ switches are common in antibody responses, and require T cell help; without it, only IgM responses are possible.
