Antibodies - Li 4/5/16 Flashcards
basic definitions
- antigen
- antibody
relationship between teh two
antigen : any substance (exog from environment or endog from within body) that generates an immune response → generation of antibodies
antibody aka immunoglobulin : protein produced by B cells that tags microbes or infected cell to either mark for immune system attack or directly neutralize
- paratope (region that binds antigen) binds epitope region on antigen
antibody structure
four polypeptide chains : 2 identical heavy, 2 identical light
- disulfide bonds connect heavy-heavy and each heavy-light
heavy and light chains have 4 and 2 domains respectively
- N terminal domains (VL and VH) are variable domains
- other domains (CL on light; CH1, CH2, CH3 on heavy) are constant domains
- heavy chain hinge domain (H) allows two arms of Ig to move relative to each other
diversity of Ig molecules
due to variability of V domains
variable domains are formed by antigen-binding (aka “combining”) sites
- comprised of framework regions and 3 CDRs (complementarity-determining regions)
- CDRs: loops of polypep at ends of paired VH and VL domains, aka hypervariable regions
- framework regions: mostly beta sheet, hold CDRs in place
- CDRs of VH and VL make up the combining site; held together by interactions between CH1 and CL domains
paratope-epitope binding
what happens with large antigens?
- each Ig has 2 identical paratopes (comprising one heavy + one light chain)
- epitopes (aka antigenic determinant) typically have 15-25 a.a. side chains that form approx 50-200 interactions with the antibody paratope
- paratopes and epitopes are s_terically and chemically complementary via multple noncovalent bonds_
- small mol? pit. long mol? groove. larger surface? lumpy surface with chemical complementarity.
large complex antigens have many epitopes
- each Ig is epitope-specific, but the population of Ig as a whole can bind the many epitopes on an antigen → process of antibody production in response to a complex antigen = polyclonal response
- best binding epitopes may direct bulk of antibody production → known as immunodominant epitopes
protease cleavage of antibodies
- what happens
- fragments produced
proteases cleave antibodies at specific sites, produce…
Fab : univalent antigen-binding gragments (1 Ig light chain + amino terminal part of 1 Ig heavy chain, linked by disulfide bond)
- contains variable part of Ig, which contains paratope and first constant region
Fc : crystalizable fragments made of carboxy-term parts of heavy chain constant regions of heavy chains, linked by disulfide bonds
papain
protease
antibody → 2 Fab fragments + 1 Fc fragment
pepsin
protease
antibody → 1 (Fab’)2 fragment + fragments of heavy chain
- (Fab’)2 includes 2 light chains and parts of 2 heavy chains, incl the hinge region
Ig gene complexes
- location
- contents
gene complexes for Ig chains occur in segments
- heavy chain genes in chr14q32.3
- light chain genes in chr2p11.2, 22q11.2
each segment has multiple versions of constant (CH), joint (JH), diversity (D), and variable (VH), some fxal and some pseudogenes
*Ig light chain → either kappa or lambda chain
somatic recombination of Ig genes
humans can make 1000B variants of antibodies, but only have 30k genes…how?
V(D)J recombination
during devpt of B cells, H and L chain genes are assembled from libraries of shorter gene segments (germ-line configuration → cutting out lots of DNA to form B cells that produce antibodies of a specific idiotype)
TCRs resemble antibody Fab regions → TCR genes are assembled by same mechanism!
- alpha chain : V and J segments
- beta chain V, D, J segments
V(D)J recombo specifics
- where/how splicing takes place
- proteins that make it happen
conserved sequences define DNA cut/splice sites
- conserved heptamer and nonamer, with spacing of specific length
- V-23bp…12bp-D-12bp…23bp-J
- splicing machiner requires 12bp and 23bp spacers in order to make cut → precludes splicing of V directly to J!
V/D and D/J splice joints occur in CDR3 region → brings diversity straight to the combining sites!
RAG1 and RAG2 (Recombo of Antibody Genes) are found only in lympocytes, possible bacterial ancestry
- RAG1/RAG2 gene pdts recognize conserved seqs and target initial cuts
proteins resp for splicing are in all cells, part of DNA repair machinery
- some DNA-repair defs can lead to immunodef!
creating additional diversity in Ig gene formation
(D)
V/D and D/J splice joints occur in CDR3 region → brings diversity straight to the combining sites!
- DNA terminal deoxyribonucleotidyl transferase (TdT, “terminal transferase”)
randomly adds nucleotides to cut ends of V, D, J segments
- TdT only found in developing lymphocytes!
affinity maturation
repeated exposure to antigen results in progressive increase in antibody affinity
- booster shots! improve affinity of antibody for antigen
basis: in germinal centers, there’s lots of somatic mutation of variable regions of Ig genes
- mutation is random…
- some will lower affinity for antigen → cells will die
- some will increase affinity for antigen → proliferate extensively, forming increasing fraction of pop
- net effect: average antibody affinity rises (low affinity die, high affinity prolif)
consequences of errant antibody gene rearrangement
VDJ rearrangement gone wrong can join VDJ region to somewhere other than constant region (i.e. another gene or another chromosome)
- this can place the joined gene under powervul B-cell specific transcriptional enhancer, which is near VDJ region…
- if the gene is involved in control of cell prolif → uncontrolled cell division, formation of clone of malignant cells :(
ex. Burkitt lymphoma
5 classes of Ig
subclasses
heavy chain defines the class/isotype of antibody
- IgA, IgG, IgM, IgE, IgD
each have distinct biological props
subclasses - determined by diffs between heavy chains
IgG has 4 subclasses
IgA has 2 subclasses
monoclonal antibodies
recognize a single epitope
- each ab producing cells makes only one species of heavy chain and one species of light chain → monoclonal
therefore, a complex antigen generates a polyclonal response
- might end up with lower specificity with a mixture of antibodies, since the likelihood of at least one epitope being found on another molecule is higher
