Marion Immunology 3

Comments:
Recommended reading is in text Immunobiology in library on reserve. AM lecture- a lot of factual information was presented. We are not expected to remember all the facts, but come away with realization that there are specific receptors to recognize different types of molecules on pathogen surfaces or RNA and DNA. For signalling, remember that the end result is activation of NFkB and MAP(?) transcription factors.Also there is common adaptor factor MyD88. Study the concepts, not minute details.

This lecture: Adaptive immune receptors and their structure and specificities. Tomorrow: genetic mechanisms.
Structure of monomeric immunoglobulin IgG: dimer of a heterodimer. Heavy chains vary (50-70 kD), light chains 25kD. Heterodimers are covalently bound by disulfide bond. One or 2-3 disulfide bonds in region called the hinge region hold it together.
We can divide the molecule into 2 distinct regions:
C or constant region provides for biological function (for cell surface and protein-protein interactions).
V or variable region provides for antigen binding.
Mid 1950s, after discovery DNA, Burnet, based on work of Medawar, proposed clonal selection hypothesis.It says that the potential for immune responses exists and develops in the absence of knowledge of what the antigen would be. Diversity is built into the system generating the receptors. Antibodies are formed prior to exposure to antigen. People who opposed this theory were instructionists. They proposed that cells took in antigen and wrapped protein around it as template and spit it out. That was shot down well by work from Dreyer and Bennett in 1965. They proposed from protein sequencing of IgGs that about 1/3 of heavy chain and 1/3 of light chain had variable sequence from one chain to another. They examined myeloma proteins. A given tumor cell produced only one type. Other part was constant. Allelic differences within the constant region were inherited as polymorphisms. Hypothesized that for Igs, 2 genes encoded one polypeptide. They were right. Alternative theory was mutational theory. It said that there was one variable region gene that was mutated by B cells. That isn’t how B cells develop the receptor they start with.
Basic building block of antibody molecule is immunoglobulin domain. Light chains have 2 domains, 1 V and 1 C. Heavy chains, some have 4 (1 V, 3 C), some 5(1 V, 4C). Of all proteins encoded by human genome, the Ig domain is most represented. Why? Stability. Among protein structures, this is most stable. Why? See slide 1. 2 parallel beta sheets disulfide linked. Formed of antiparallel strands connected by alpha helical loops. These are very stable.Structures of Ig domains in proteins differ, but the function depends on the overall structural stability.
Variability is in alpha helical loops.
The constant region of IgM and IgG is formed from 3-4 constant region Ig domains.
IgGs-3 CH1, CH2, CH3. Primary structure is different, but secondary structure is the same.
variable domain- sequence varies, but amount of variability differs from one part to another.
Slide 2:Wu and Kabat were early investigators of Ig structure. Took all known Ig sequences, aligned to study, looked for relationships one to another. Compared. Constant regions were the same, but variable regions had periodicity. Some parts have much less variability than others. “Framework” regions represent B strands. Structural integrity is maintained by relatively constant sequence.
Variable regions can be divided into 10-12 families in humans. Red on graph- complementarity determining regions- CDRs. Also called hypervariable regions. These are parts of molecule contacting the epitope.
Epitope- the molecular determinant of antigen (Ag)to which antibody(Ab) binds.
antigen- something that an antibody or T cell receptor (TCR) can bind.
immunogen- antigen that can induce an immune response. All antigens are not immunogens. There are some things an antibody can bind that are not immunogenic.
Igs have 5 classes, depending on the C region structure.
IgM
IgG
IgA
IgE
IgD
All found in circulation except for D. D is never secreted. Found on D cells.
Classes also referred to as isotypes. Isotype explains antigenicity of constant region. Can immunize mouse with human IgG. Isotypes are mu, gamma, alpha, epsilon, delta. Refers to heavy chain for each class. Two isotypes light chain: kappa and lambda.
allotypes: 2 of each for heavy and light chains.
One other type of antigenic determinant: idiotype refers to the structure created by heavy and light chain variable regions. There are idiotypic differences from one molecule to another.
In addition to 5 classes, IgG has subclasses 1,2,3,4. IgA has 1 and 2.
Mouse IgG subclasses IgG 1, 2a, 2b, 3. identified on the basis of structural variation in variable regions. Different subclasses dominate different immune responses depending on the antigen stimulating the response.
IgM and IgA are secreted in polymeric form (IgA dimeric, IgM pentamer). Plasma cell secretes J polypeptide, which disulfide binds 2 IgM monomers to polymerize. Creates 5 IgM monomers bound in constant region. For IgA, J forms dimer in mucosal secretions only.
Beginning in 1940s and 50s first studies of Ig structure were performed by proteolysis by papain and pepsin. ONLY relevant to IgG. Papain cleaves molecule into 3 frags: Fab- antigen binding. Fc-crystallizable. Pepsin forms F(ab’)2 and peptides.
F(ab’)2 could agglutinate or aggregate. Could act like intact antibody. Monomeric IgG had to be a dimer of a heterodimer.
How antibodies bind to antigens:
binding is noncovalent, kinetic interaction. KA can be measured by equilibrium dialysis. KA is dependent on association and dissociation. Referred to as affinity of binding an antibody. Measures interaction of a single Fab and one Ag. Affinity of an antibody molecule is always higher because they may be dimers to pentamers. Avidity is relative affinity that is consequence of its valence (number of monomers together) and its true affinity. When you measure interactions, single interaction is independent to a point, depending on how many determinants are on the molecule. Multiple epitopes increase the apparent affinity. When one of the interactors is immobilized to a solid phase, interaction is changed to first order reaction and changes relative affinity by as much as four logs.
T cell receptors:
Essentially antibody Fab. Found membrane bound, and only on T cells. 2 polypeptides make the receptors:
alpha and beta.Alpha is like the Ig light chain. B is like the heavy chain structurally. Had V and C regions. Variability in V region allows recognition of antigens.
Antibody and B cell receptor can bind native antigen. That means antibody can bind protein or carbohydrate which does not have to be changed for it to bind.
T cell receptors don’t do that. they are selected for their ability to bind peptide in MHC molecule.
MHC-Major histocompatibility complex. these molecules are detected as transplantation antigens. MHC encoded molecules generate the strongest transplantation responses.
2 types MHC:
Class I
Class II
Both bind peptides derived from proteins proteolytically digested in antigen presenting cells (APCs). APCs are cells that proteolytically process proteins into peptides bound by MHC. All nucleated cells make class I MHC. Only a subset (Professional APCs) express class II (macrophages, dendritic cells, B cells). Allows peptides from antigens inside and outside the cell to be expressed inside one of these to be sampled. TcR is selected to bind only to peptides in Class I or II MHC. T cell does not do a thing to virus or bacterium- recognizes infected or tumor cells.
Depending on type of T cell, immune system divides and conquers. CD4 T cells class II, CD8 in class I. Selection for specificity happens in thymus.
Slide 6: Pockets, grooves, or planar surfaces can be formed by heavy and light chains. interactions and affinities are electrostatic, hydrophobic,e tc.
MHC restriction- Peter dougherty discovered this concept: T cells only bind to antigens bound to MHC class one or II. If MHC wrong- no binding. Wrong peptide- no binding.
12: T cell receptors interacting with peptide and Class II MHC.
13: Ig is co-expressed with Ig alpha and beta, which engage sark like kinases. receptor functions to bind.
T cell receptor coexpressed with CD3. Zeta is signal for T cell receptor. ITAM phosphorylated, attract kinases to initiate signalling.