B3.061 Proteins in Immunity Flashcards
what are the major protein classes that are players in immunity?
Immunoglobulins/ Antibodies
MHC complexes/ TCRs
Lectins
which Ig classes are monomers?
IgD, IgE, IgG
which Ig class is a dimer
IgA
which Ig class is a pentamer
IgM
what makes up the binding sites of an immunoglobulin?
dynamic loops
Velcro-like mechanism
effective at binding a wide range of antigens
where are Ig binding sites on the Ig?
Fab regions (top parts of the Y shape)
what are some examples of antigens that can be bound by Igs?
whole proteins peptides DNA/RNA carbs haptens
what is the Fc region of an Ig
the stem of the Y shape
what can bind to the Fc region of an Ig?
Fc receptors
complement
bacterial protein A (IgG)
other species antibodies (ELISA)
when does bacterial protein A bind to the Fc region of an Ig?
when it is fighting back against the immune system
how do we design ELISA tests?
raise Abs against the Fc region of human Igs to mark them
what is located at the bottom of the BCR
C-terminal transmembrane domain
discuss the physical structure of the MHC class 1 complex
heterodimer
alpha 1, 2, 3 and B microglobulin
what does MHC class 1 bind
cytosolic antigens
8-10 aa in length
discuss the physical structure of the MHC class 2 complex
heterodimer
B 1,2 and alpha 1,2
what does MHC class 2 bind
extracellular antigens after phagocytosis
15-24 aa in length
how are peptide antigens generated?
by the proteasome
takes in self and mutant/viral proteins
spits out chunks of proteins that are them displayed by class 1 MHC on cell surface
what part of the TCR interacts with an antigen/MHC complex?
complementarity determining region (CDR) loops
TCR binds composite surface of peptide and MHC protein
discuss the physical structure of the TCR
heterodimer a and B chains V region, C region, hinge, transmembrane region (top to bottom) coated with carbohydrates on outside chains linked by disulfide bond
how does TCR discriminate MHC1/2?
CD4/8 co-receptors
what are lectins?
receptors that recognize carbohydrates
who expresses lectins?
hosts, microbes, plant toxins
allows for a wide range of binding combos
the carb or the lectin can be on either type of cell
describe the structure of lectins
extremely variable
grouped into 6 families
can be grouped into higher order structures with multiple binding sites
what are the 4 characteristic that are needed in an immune response
diversity
specificity
high affinity
stability
diversity
body must be ready to encounter any of 1000s of antigens
specificity
discrimination among potential antigens
high affinity
must bind tightly to antigen (time vs equilibrium)
must be stable and persist
stability
must survive in circulation or on the cell surface
what are the 2 different options for protein engineering diversity
option 1: make thousands of different proteins
option 2: a few proteins bind many ligands
**either way must be ready to encounter thousands of antigens
how do Igs and TCRs achieve diversity?
option 1
combinatorial power
-VDJ gene recombination
-high frequency somatic mutations (only Igs)
throw out self and amplify those that encounter antigens
how is alternative splicing in DNA utilized in Ig heavy chain?
as B cells mature, they start rearranging and changing their DNA sequence
this process is deliberately error prone with varied domain boundaries and random insertions, deletions
lists the steps in Ig VDJ recombination within DNA
- all genes in heavy chain locus
- removal of unwanted D and J gene segments
- recombination of D and J exons (DJ recombination)
- removal of unwanted V and D gene segment
list the steps in Ig VDJ recombination done at the mRNA level
- recombination of V and DJ exons
antibody transcript will also include constant domain gene
what is the function of alternative splicing in the mRNA utilized in the Ig heavy chain?
allows isotype switching while maintaining antigen specificity chosen
chosen C region can code the membrane or the secreted form
how do MHCs achieve diversity?
option 2
more diversity across the human population than within an individual
genetic cross over during meiosis leads to these polymorphisms
most polymorphic genes known
MHC
every person possesses a set of MHC molecules with different ranges of peptide binding specificities
evolutionary insurance??
MHC specificity
broad
how do MHC 1 molecules bind peptides
peptide stabilized by anchors at both ends
how do MHC 2 molecules bind peptides
peptide side chains protrude into pockets lined by polymorphic residues more permissive than class 1 no clear binding rules
TCR/Ig specificity
high/narrow
each variant has a reasonably strict specificity
can you predict a TCR/Ig’s structure from its sequence?
nope, makes it hard to make targeted drugs
what is an example of cross reactivity leading to disease?
lonestar tick induced allergy to red meat
body develops an immune response against ticks
similar antigen present in meat which is then recognized by immune system following tick exposure
how can drugs mediate self vs non self mistakes?
HIV drug abacivir
drug binds to self peptide leading to a ‘new’ self peptide that is not recognized as self
causes and immune response
what proteins have some of the highest protein-protein binding affinities?
antibodies
TCRs
nearly permanent
how does lectin-carb binding affinity compare to antibodies/TCR?
relatively transient
how many biding sites are on 1 Ab?
2 identical and independent binding sites
do the 2 Ab binding sites act completely independently?
no
cooperativity arises through protein communication
discuss the concept of avidity
more binding sites increases avidity
after the first binding event, subsequent binding events have high local concentration
making binding affinity higher than a single Ab-Ag affinity would be
discuss the binding affinity of IgM molecules and the overall avidity
IgM antibodies are produced before B cells undergo hypermutation
tend to have lower affinity than other isotypes
10 Ag binding sites can bind simultaneously to multivalent antigens
high overall avidity
discuss the differences in complement activation by IgM and IgG
only 1 IgM required to activate
at least 2 IgGs required
what does multi-valency allow for?
clustering
different cluster formations can send different signals
why is having a high abundance of proteins important?
hard to inhibit
Abs : 18% of total blood protein
TCR: 30,000 on T cell surface
cell surface carbs: clusters measured in um, there are so many
why is delivering drugs to target specific microbe receptors so difficult? i.e. flu hemagglutinin
need a HUGE concentration in body to have an effect
what are 2 key factors that provide protein stability?
glycosylation
disulfide bonds
where is the Ig molecule glycosylated
in the middle of the Fc region
how does the glycosylation act on the Ig
covalent osmolyte
helps the molecule to stay folded and block proteases
how are antibody drugs designed?
de novo design doesn’t work (cant design an Ab for a specific Ag)
make a huge library and screen for the one that works
discuss the stability requirements of Ab drugs
must survive on the shelf (refrigeration) delivery mode (infusion, don't survive GI tract) solubility (concentrated drug gets diluted in vivo, so has to be super concentrated at administration to avoid volume overload)
how are humanized antibodies used?
antibody template from some other organism
constant regions/carb altered to resemble human
diminishes chance of immune response to antibody drug
