Introduction to the Immune System 3. Flashcards
What are antibodies composed of?
They are Y shaped molecules copses of 2 light chains and 2 heavy chains
How heavy are the heavy chains that make up antibodies?
50 kDa
How heavy are the light chains that make up antibodies?
25 kDa
What hold the 4 polypeptide chains together in antibodies?
Disulphide bonds
What specifically do the disulphide bonds link in antibodies?
2 disulphide bonds link the heavy chains
1 disulphide bond links each light chain to its heavy chain partner
So 4 disulphide bonds in total
What are the 2 main groups of functions antibodies have?
- To recognise and bind antigens
2. To elicit effector functions
Name the 2 different regions of the antibody
- The variable region
2. The constant region
What function does the variable region complete?
It provides the antigen binding function of the antibody
What function does the constant region complete?
It elicits the effector function (eg recruits additional immune cells OR cells to destroy pathogens following antigen binding)
Where are the variable region found?
At the amino (end) terminals of the polypeptides
Where are the constant region found?
At the C terminus
Define affinity
The strength of binding of one molecule to another at a single site
Give an example of an event that shows affinity
The binding of a monovalent Fab fragment of antibody to a monovalent antigen
Define avidity
The sum of the strength of bonding of 2 molecules or cells to one another at multiple sites
Antibodies are bifunctional. What does this mean?
It means they have 2 functions:
- To recognise and bind antigens
- To elicit effector functions
How are antibodies digested?
By different proteases
What are proteases
An enzyme that cut up proteins
What has the digestion of antibodies allowed us to do?
Dissect antibodies an be able to view them at a molecular level to see what bit does what function
Name the 2 enzymes that perform important digestive functions
- Papain
2. Pepsin
What does papain do?
It cleaves the antibody to produce 2 Fab fragments and 1 Fc fragment
What does a Fab fragment do?
It recognises the antigen
Why is the Fc fragment given this name?
It is the easiest fragment to crystallises
fragment crytaliseable
Why is the Fab fragment given this name?
Means fragment antibody binding
What does an Fc fragment do?
It binds to cell receptors to recruit other ells of the immune system
What does pepsin do?
It cuts the antibody to give a F(ab’)2 fragment
Describe the F(ab’)2 fragment
It has 2 antigen binding sites as the 2 Fab arms are still linked together
Do the Fab and F(ab’)2 have the same AFFINITY for antibodies?
Yes it is the same
Do the Fab and F(ab’)2 have the same AVIDITY for antibodies?
No it is different
What are the 2 types of light chains found in humans?
- Kappa (k)
2. Lambda (λ)
Which type of light chains do each antibodies have?
Any particular antibody has either 2 K chains or 2 λ chains
never one of each
What is the average ratio of B cells prosecuting kappa light chained antibodies to lambda?
2:1
Name a situation where the ratio of B cells prosecuting kappa light chained antibodies to lambda?may be skewed?
If someone were to have cancer
What are the 5 different classes of antibodies called?
- IgG
- IgA
- IgM
- IgD
- IgE
Which heavy chains make up IgG?
Gamma chain
Which heavy chains make up IgA?
Alpha chain
Which heavy chains make up IgM?
Mui chain
Which heavy chains make up IgD?
Delta chain
Which heavy chains make up IgE?
Exelon
What 2 forms can IgA take?
The monomeric
The polymeric form (a diamer)
What 2 forms can IgM take?
The monomeric
The polymeric form (a pentamer)
What do both heavy and light chains contain?
A repeated similar domain called the immunoglobulin fold
How may immunoglobulin folds are there in IgG light chains?
2
How may immunoglobulin folds are there in IgG heavy chains?
4
Where is all variability in the antibody found?
At the amino (N) terminal domains of the light and heavy chains
Give some other examples of cells that have immunoglobulin folds but are not immunoglobulins?
- T cell receptors
- MHC
- CD5
Where is the antigen binding site found?
At the amino (N) terminal domains of the light and heavy chains (the variable regions)
What is each immunoglobulin fold composed of?
2 antiparallel beta pleated sheet held together by disulphide bonds?
What are beta strands linked by?
Flexible loops
What do the flexible loops linking beta strands allow?
Allow strands to alternate direction
What is amino acid sequence variability concentrated in?
In the hypervariable regions or Complementarity Determining Regions (CDRs)
What is located at the tip of each Fab arm?
3 CDRs in the heavy chain and 3 CDRS in the light chain
What dies CDR stand for?
Complementarity Determining Regions
What do the 6 CRDs at the tip of each Fab arm combine to generate?
Combine to generate antigen binding sites
How do changes in amino acid sequences lead to changes in antigen binding specificity?
Changes in amino acid sequence at hypervariable regions alter the Complementarity Determining Regions which in turn leads to changes in antigen binding specificity
What is Combinatorial diversity ?
Different specificities created by different combinations of heavy and light chains.
Antigen and antibody bind via non-covalent interactions which can be disrupted by what?
- High salt concentrations
- Extreme pH
- Detergents
- High concentrations of purified epitope
Name some of the non-covalent interactions which can bind Antigen and antibody?
1. Electrostatic forces 2. Hydrogen bonds 3. Hydrophobic forces 4. Van der Waals forces
Where are Electrostatic
forces found?
Between oppositely charged amino acid side chains on antibody and antigen
What can disturb electrostatic forces?
High salt
Where are Hydrogen bonds found?
When Hydrogen is shared between negatively charged atoms.
What can disturb hydrogen bonds?
High salt
Where are hydrophobic
forces found?
Found when Hydrophobic groups pack together, excluding water
Has a short range
Where are Van Der Waals
forces found?
Where fluctuations in electron clouds occur leading to opposite polarisations in neighbouring atoms.
What are antibodies used in?
Diagnostics
Research
In drugs
What are the functions of different Ig classes determined by?
The Fc regions
Gives some ways antibodies complete their function
- Neutralisation
- Opsonisation
- Activation of the complement pathway to act as opsonin or lysis.
What is neutralisation
Antibodies bind to pathogen and toxins, and prevent binding to the cellular receptors pathogens used to gain entry to cells.
What is Opsonisation?
The coating of pathogen with antibody
This results in phagocytes recognising the Fc region of that pathogen causing the phagocyte to engulf the pathogen
Where are antibodies present
In a lot of different places in our body: Much Lining airway Serum Fluid that bathes our tissues
Describe the process of neutralisation
- Antibody blocks the binding of microbes to cells
- Antibody clocks the infection of adjacent cells
- Antibody clocks the blinding of toxin to cellular receptor
What is the newest potential role of antibodies?
If an antibody bound to a virus is taken up by a cells an intracellular receptor called TRIM21 detects the antibody inside the cell and recruits the proteasome machinery
What do proteasome do inside the cell?
The proteasome degrades the antibody virus complex.
Where is TRIM21 found and what is it?
It is an enzyme found inside cells
Name where our central (primary) lymphoid organs are found?
- Thymus (T cells)
2. Bone marrow (B cells)
Name where our Peripheral (secondary) lymphoidal organs are found?
- The adenoids
- Tonsils
- Lymph nodes
- Spleen
- Peyer’s patches
- Appendix
Define Antibody repertoire
The total range of antibody specificity
What is the estimated Antibody repertoire ?
1 x 10^11
Why is a diverse Antibody repertoire required?
required to recognise vast array of more rapidly evolving pathogens.
what do dendritic cells do
Cells that have taken up antigens from the site of infection to the lymph nodes in order to display the antigens to keep the immune repose going
What is the estimated total number of lymphocytes in the body?
4x10^11 and 2x10^12,
Where does variation in Ig derive from?
Ig variation is derived from changes in the amino acid sequences which occur within the CDRs, altering the antigen binding sites.
What is the problems with great Ig diversity?
Theres not enough genomic space
What is the solution our body has to the lack of genomic space to code all the different Ig variations
Somatic Recombination.
What have Ig genes in B cells been rearrange by?
Somatic Recombination.
What are gene segments?
Variable regions of Ig genes are encoded in multiple pieces
What is germline diversity?
Multiple choices for each gene segment
What provides combinational diversity?
The process where by Segments are randomly selected and brought together in different combinations
The variable region in light chains are encoded by what segments?
2 types of segment:
Variable (V) and Joining (J)
The variable region in heavy chains are encoded by what segments?
3 types of segment:
Variable (V), Diversity(D) and Joining (J)
Name the 3 sets of immunoglobulin chains
- Kappa light chain
- Lambda light chain
- Heavy light chain
What MUST genes encoding the variable region be present in?
Must be present as multiple gene segment
Name the 3 immunoglobulin loci
- Kappa chain locus
- Lambda chain locus
- Heavy chain locus
Where is the Kappa chain locus present?
Chromsome 2
Where is the lambda chain locus present?
Chromsome 22
Where is the heavy chain locus present?
chromosome 14
At the 5 prime end of each locus what is present?
A cluster of V (variable) gene segments
What is each V segment associated with?
A leader (L) sequence
What does the L sequence encode?
It encodes a signal or leader peptide that facilitates the transport of Ig protein through the endoplasmic reticulum
Is the leader sequence present in mature Ig?
No it is cleaved before hand
At the 3 prime end of each locus what is present?
A cluster of J (Joining) gene segments
What do J gene segments code for?
The portions of variable region of Ig chains
How are the 2 segments in light chains ( the variable and the joining) brought together?
They are Brought together by one somatic recombination event
Describe the process of Light chain gene rearrangement.
1 .Gene rearrangement of V and J occurs via one somatic recombination event to give an exon
- The Gene is transcribed
- The remaining introns are removed during RNA splicing
- The leader sequence is translated targeting protein for secretion or expression on the cell surface.
- Leader sequence is cleaved off post translation
What is V (D) J recombination?
A process by which B cells randomly assemble immunoglobulin gene segments (ie v, d, j gene segments) to form the function variable region exon
When does V (D) J recombination occur?
During early development of lymphocytes
What does somatic DNA recombination do during Light chain gene rearrangement?
The gene segments are rearranged to form a complete variable region coding sequence
Give one word or phrase to describe each step in the process of Light chain gene rearrangement.
- somatic DNA recombination
- Transcription
- Splicing
- Translation
- Cleaving
Describe the process of Heavy chain gene rearrangement.
- Gene rearrangement of D J via a somatic recombination event
- ANOTHER gene rearrangement occurs of V-DJ via a somatic recombination event to give a complete exon
- The Gene is transcribed
- The remaining introns are removed during RNA splicing
- The leader sequence is translated targeting protein for secretion or expression on the cell surface.
6 . Leader sequence is cleaved off post translation
What is a key difference between light and heavy chain rearrangement?
In light chain rearrangement there is only 1 gene rearrangement event (V-J joining)
In HEAVY chain rearrangement there are TWO gene rearrangement events (V-J joining AND V-DJ joining)
What controls the V(D)J recombination?
Controlled by non-encoding, conserved DNA sequences
Recombination Signal Sequences (RSS)
What does a Complete functional rearrangement require?
One of each type of segment is joined in the correct order and in the correct transcriptional orientation
Where can recombination only occur?
Recombination can only occur between one gene segment flanked by a 12 spaced RSS and another with a 23 spaced RSS.
How Many types of RSS are there?
2:
One with 23 base pair spacer
One with a 12 base pair spacer
Approx how many V segments do we have in our heavy chains?
45
Approx how many V segments do we have in our kappa light chains?
35
Approx how many V segments do we have in our Lambda light chains?
30
Approx how many D segments do we have in our heavy chains?
23
Approx how many J segments do we have in our heavy chains?
6
Approx how many J segments do we have in our kappa light chains?
5
Approx how many J segments do we have in our lambda light chains?
4
Why do the number of segments in each Ig vary in the population?
Because humans are very polymorphic
Approx how many heavy chain combinations are there?
V x D x J
45 x 23 x 6
=6000
Approx how many kappa light chain combinations are there?
V x J
35 x 5
= 175
Approx how many lambda light chain combinations are there?
V x J
30 x 4
= 120
Approx how many light chain combinations are there IN TOTAL?
potential kappa + potential lambda
175+120= 295
Why are the number of potential light and heavy chains number very varied?
Because segment numbers are varied
Some combinations produce non-functional pseudogenes
What is the third level of diversity called (after gremlin and combinatorial)?
Junctional diversity
When does junctional diversity occur?
When selected gene segments have to be joined together
Describe how junctional diversity is achieved
- RAG proteins cleave the DNA.
- Nucleotides are added or subtracted to make a viable joint by TdT (terminal deoxynucelotidyl transferase).
- The position of the cut adds P nucleotides to the sequence, whilst the TDT adds N nucleotides.
What does RAG stand for in RAG proteins?
Recombinase-activating genes 1 and 2
What are RAG protein responsible for?
For cutting and cleaning DNA
Is the process of achieving junctional diversity efficient?
No there’s a lot of wastage because in the process of deleting and adding nucleotides we can get frame shifts
Name the 3 layers of diversity in T cells and antibodies?
- Germline
- Combinatorial
- Junctional
What can genetic immunological disorders affect?
- Severe combined Immunodeficencies (T and B cell function)
- Deficiencies in antibody production
- Phagocytic disorders
- Complement deficiencies.
Which fragments of antibodies have the same AFFINITY?
the Fab and F(ab’)2
What hold together the 2 antiparallel beta pleated sheets in each immunoglobulin fold?
Disulphide bonds
Which fragments of antibodies have different AVIDITY?
the Fab and F(ab’)2
Gamma heavy chains make up which class of antibodies?
IgG
Alpha heavy chains make up which class of antibodies?
IgA
Mui heavy chains make up which class of antibodies?
IgM
What does the Fc region determine?
The different classes of antibodies
Delta heavy chains make up which class of antibodies?
IgD
Exelon heavy chains make up which class of antibodies?
IgE
What is the immunoglobulin fold?
A repeated similar domain found on both light and heavy chains
