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Immunology > Antigen recognition and antibody diversity > Flashcards

Antigen recognition and antibody diversity Flashcards

1
Q

how do adaptive immune responses differ innate responses?

A

Adaptive immune responses are initially slower to develop than innate responses, but show SPECIFICITY and MEMORY.

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2
Q

how do B and T cells recognise antigens?

A

through their specific antigen receptors
- antibody receptor
- TCR receptor

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3
Q

where do B and T cells acquire their antigen receptors?

A

B cell = bone marrow
T cell = thymus

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4
Q

how diverse are B and T cell antigen receptors?

A

Single B or T cells express single antigen receptors.
- Cells with appropriate receptors undergo CLONAL SELECTION in response to antigen.
- B and T cell receptors are extremely diverse (>109).

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5
Q

what are the two ways in which antibodies can exist?

A
  1. integral membrane proteins on B lymphocytes (antigen receptors)
  2. soluble proteins secreted by plasma cells (antigen eliminators) – mark foreign material for elimination by the immune system
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6
Q

what is the structure of an antibody?

A

4 polypeptide chains linked by disulphide bonds:
- 2x Light (L) chains = each 25kD
- 2x Heavy (H) chains = each 50kD
- Immunoglobulin G = L2H2 = 150kD
- Hinge region is flexible to enable Fab arms to move independently of one another to bind to antigen at different distances apart

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7
Q

what happens when antibodies are cleaved by papain?

A

yields two types of fragments:
1. Fragment antigen binding = Fab arm
- F(ab)2 fragment can bind antigen divalently, as in the intact molecule

  1. Fragment crystallisable = Fc effector region
    - interacts with elements of the innate immune system for antigen elimination
    - crystalises in solution
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8
Q

how are immunoglobulins classified?

A

by the amino acid sequence of their heavy chains

also 2 light chain types (kappa and lambda), but these are not class restricted

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9
Q

How was the structure of antibodies studied

A

the protein sequencing of large numbers of myeloma proteins showed that antibodies are made of constant and variable regions

myeloma = cancer of plasma cells - single antibody is continously produced

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10
Q

what are the 5 immunoglobulin classes?

A
  1. IgG - main class in serum and tissues - important in secondary responses (specific)
  2. IgM - important in primary responses (binds to many antigens)
  3. IgA - in serum & secretions (mucus, tears, saliva, milk) - protects mucosal surfaces
  4. IgD - unknown ?
  5. IgE - present at very low levels - involved in protection against parasites and allergy
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11
Q

what two regions do antibodies posses?

A

constant and variable regions

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12
Q

what are the variable regions of antibodies?

A
  • VARIABLE (V) regions - bind antigen.
  • Differ between antibodies with different specificities.
  • N-terminal regions of heavy and light chains
  • Forms the antigen binding site
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13
Q

what are the constant regions of antibodies?

A

CONSTANT (C) regions - same for antibodies of a given H chain class or L chain type.
- Rest of the light chains and heavy chains
- Interacts with elements of the innate immune system

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14
Q

how are antibodies comprised of homologous domains?

A
  • Regions of sequence homology identified (~110 amino acids including 2 cysteines).
  • Cysteines form intramolecular disulphide bonds
  • Hypothesized to form a series of globular domains, each stabilised by an intra-chain disulphide bond
  • Domain structure confirmed by X-ray crystallography of Fab and Fc fragments
  • Hinge region with proline residues confers flexibility, but is susceptible to proteolysis
  • The folding pattern of the domains is known as the Immunoglobulin Fold
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15
Q

what is the immunoglobulin fold?

A
  • C domain has 7 beta strands, V domain has 9 beta strands to form beta-pleated sheets
  • Beta pleated sheets stabilised by intrachain disulphide bonds
  • Beta strands are joined by loop regions
  • Found in all members of Immunoglobulin Gene Superfamily
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16
Q

what is the immunoglobulin gene superfamily?

A
  • Superfamily generated by single primordial gene
  • Generally involved in recognition, binding, adhesion
  • > 700 members in the human genome – biggest gene family in humans
  • Domains can be V-like (Ig-V) or C-like (Ig-C)
  • Loops can be modulated without disturbing the whole structure
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17
Q

how are variable regions diverse?

A

they contain hypervariable loops in the antigen-binding site

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18
Q

what are hypervariable loops?

A
  • Hypervariable regions (~7-12 amino acids) bind to antigen
  • Separated by beta-sheet framework regions
  • Hypervariable regions = Complementarity Determining Regions (CDRs)
  • Hypervariable regions correspond to the loops found at the end of the variable regions for antigen binding
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19
Q

what kind of bond is formed between an antigen and antibody? is this bond strong?

A
  • The interactions between the antibody and antigen are non-covalent (weak)
  • e.g. Electrostatic interactions, Hydrogen bonds, Van der Waals forces, Hydrophobic interactions

Individually weak, but if many form simultaneously (i.e. if the antigen bindingsite and antigen contain many COMPLEMENTARY residues), the antibody:antigen interaction is specific and of high affinity

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20
Q

which part of an antigen does the antibody bind to?

A

the epitope

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21
Q

what is the structure of B cell immunoglobulin receptors?

A
  • Membrane immunoglobulins contain ~26 hydrophobic a.a. at the C-terminus – enables receptor to sit in the membrane
  • Most B-cells express monomer IgM and/or IgD as B-cell receptors.
  • Recognise and bind to antigen, but cannot generate a signal.
  • Only a few amino acids enter the cytoplasm
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22
Q

what proteins are membrane immunoglobulins associated with?

A
  • Membrane immunoglobulins are associated with two other proteins, Ig-alpha and Ig-beta.
  • Ig-alpha and Ig-beta contain a single ITAM (Immunoreceptor Tyrosine Activation Motif) in their long cytoplasmic domains to enable some signalling
  • ITAM is phosphorylated when antigen binds to antibody, which triggers signalling
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23
Q

what is the immune repertoire of antibody receptors and TCRs?

A

~10^14 antibody receptors

~10^18 T cell receptors

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24
Q

how is antibody diversity generated by its structure?

A
  • Diversity is based on variable and hypervariable regions
  • Variations in the sequence and length of CDRs are the main determinants of antibody diversity.
  • CDR3 tends to be most variable in length and sequence
  • Heavy chain generally contributes more to antigen binding and is more variable than the light chain
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25
Q

how does antibody diversity arise genetically?

A

Diversity arises by somatic recombination and mutation of a limited number of inherited gene segments, which make up the variable regions.

this enables generation of 10^14 antibody repertoire

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26
Q

what were the early hypotheses of generation of antibody diversity?

A
  1. multiple genes
  2. somatic mutation in B cells
  3. somatic recombination

1965 - immunoglobulins are encoded by a separate C region and multiple V region genes

1976:
- Immunoglobulin genes are rearranged during B cell development
- Single constant region gene and multiple variable region genes in the germline
- When B cells differentiate, the DNA rearranges, so that a particular variable region gene is adjacent to the constant region gene to encode a light chain

27
Q

how was it demonstrated that B cell genes recombine?

A
  • Embryo pattern (germline) found in all cells except those of B lineage (myeloma cells)
  • V and C region genes had been rearranged to be close together in B cells
  • Only B cells undergo this process

DNA was extracted and digested with restriction enzymes
restriction fragments were separated by electrophoresis
V and C regions identified by hybridisation with probes

28
Q

what are the 3 sets of immunoglobulin genes? what gene locus do these have?

A
  1. Set coding for Heavy (H) chains found on chromosome 14
  2. Kappa chains – chromosome 2
  3. Lambda chains – chromosome 22
29
Q

what does an immunoglobulin gene locus contain?

A

Each locus has multiple VARIABLE region genes and one, or a few, CONSTANT region genes

30
Q

how many exons encode variable regions and constant regions?

A

The variable regions are encoded by 2 or more exons

Constant regions are encoded by 1 exon

31
Q

how many segments of DNA are light chain variable regions encoded by?

A

Light chain V regions are encoded by 2 segments of DNA:
- Kappa chains have a single C gene and multiple genes downstream from this that form the variable region
- Most variable region is encoded by V-kappa, and the rest by J-kappa
- J stands for joining region

32
Q

how many segments of DNA encode heavy chain variable regions?

A

Heavy chain V regions are encoded by 3 segments of DNA:
- 3 exons: V gene, D (diversity) gene, J gene

33
Q

what immunoglobulin heavy chain is expressed first?

A

IgM

34
Q

how many V, D and J segments are present in the immunoglobulin chains?

A

The V, J and D segments are present in multiple copies in the genome
For kappa chains:
- 1 constant region gene, 5 J exons and 38 V exons

For heavy chains:
- 1 constant region gene, 6 J exons, 23 D exons and 40 V exons

Rearrangement of these genes is needed so that the V segments come in close proximity to the constant region genes

35
Q

when does somatic recombination occur?

A

Rearrangement of light and heavy chain genes occurs during B cell differentiation
- permanent changes in the DNA

36
Q

how does light chain recombination occur?

A
  • a V gene is spliced to a J gene and the intervening DNA is excised (V2 and J4 are now close)
  • Rearranged V promoter is now close to enhancer, allowing transcription
  • Intervening sequences are removed by RNA processing/splicing
  • Produces mRNA with constant region and variable region
37
Q

how does heavy chain recombination occur?

A
  • D segment rearranges to lie next to J segment: DJ joining
  • V gene is somatically recombined with the DJ segment: VDJ joining
  • Unwanted parts of transcript are removed via RNA processing to produce mRNA with D and J exons between the variable and constant regions
38
Q

how are the CDRs encoded?

A
  • CDR1 and CDR2 are encoded by the V segments (i.e. germline)
  • CDR3 corresponds to the VDJ (or VJ for light chain) join
39
Q

what is the mechanism of somatic recombination?

A

Involves lymphocyte-specific recombinases and conserved recognition signal sequences (RSSs):
- RSS = conserved heptamer (7 b.p.) + nonamer (9 b.p.) separated by 12 or 23 random nucleotides
- RSSs are found directly adjacent to the coding sequence of V, D or J gene segments. These guide rearrangement of the V, D and J segments.

40
Q

what is the 12-23 base pair rule of somatic recombination?

A

12-23 base pair rule:
- a gene segment with a 12 bp spacer only joins with a gene segment with a 23 bp spacer.
- Ensures correct V-D-J joining

41
Q

what is V(D)J recombinase?

A

Complex of several enzymes required for somatic V-region gene recombination:
- normal DNA cleavage/repair enzymes
- RAG1-RAG2 protein complex (encoded by Recombination Activation Genes) - specialised endonuclease expressed only in developing lymphocytes
- terminal deoxynucleotide transferase (TdT)

42
Q

what happens when RAG genes are muated?

A

Mutations in RAG genes result in severe combined immunodeficiency (SCID)

43
Q

how does the V(D)J recombinase function during somatic recombination?

A
  • During recombination, the RAG1-RAG2 complex recognises and aligns the RSSs adjacent to the gene segments to be joined
  • The RAG1-RAG2 complex has endonuclease activity and cleaves the DNA
  • The cleaved DNA is repaired to form the coding joint (V and J segments now next to one another) and the signal joint (intervening DNA excised
44
Q

what is the structure of RAG1/RAG2?

A
  • Structure of RAG1-RAG2 complexed with DNA explains the 12-23bp rule.
  • Dimer of RAG1 which recognises the DNA is associated with RAG2 dimer

RAG1 has 2 domains:
- NBD domain = nonamer binding domain
HMGB1 = high mobility group box 1 protein

45
Q

how does the conformation od RAG1-RAG2 change when binding to DNA?

A
  • When it isn’t binding DNA, it has an open configuration which closes when bound
  • When it binds DNA, it bends towards 12-RSS sequence so that the other side is forced to bind a 23-RSS sequence
  • HMGB1 facilitates bending
46
Q

what is the function of RAG1-RAG2?

A
  • RAG1-RAG2 effectively acts as a transposase to drive somatic recombination
  • Essential in the development of adaptive immunity
47
Q

why is somatic recombination imprecise?

A

due to junctional diversity

48
Q

what is junctional diversity? how does it aid antibody diversity?

A
  • Nucleotides may lost or added: variable addition of nucleotides at junctions contributes to diversity of CDR3
  • Terminal deoxynucleotide transferase (TdT) randomly adds up to 12 nucleotides to V-D-J joins (heavy chain only)

Junctional diversity increases overall diversity by a factor of 3 x 10^7

49
Q

what is the step-by-step process of somatic recombination?

A
  1. RAG-1-RAG-2 complex recognises and aligns the RSSs adjacent to the gene segments to be joined (germline DNA folded)
  2. RAG1 introduces 2 ssDNA breaks are made close to the RSSs to expose a OH group
  3. Free 3’-OH attacks phosphodiester bond on other DNA strand to create a hairpin at the segments to be joined and a flush double-strand break at RSS boundary.
  4. 7 other proteins bind to repair the joints, but this process is imprecise, with nucleotides added or subtracted.
    - DNA hairpins are cleaved at random, symmetrically
  5. or asymmetrically (5) – palindromic overhang
  6. For V-D-J joining of the H chain, nucleotides can be added by terminal deoxynucleotide transferase (TdT)
  7. Unpaired overhangs are filled in by DNA polymerase or may be excised by an exonuclease.
  8. DNA ligase joins the nicked and repaired hairpins to form the “coding joint”. (The blunt ends formed at Stage 3 are ligated to form the “signal joint” and this DNA is typically excised)
50
Q

how much diversity does heavy and light chain recombination provide?

A

3.3x10^6 repertoire

51
Q

what are the 4 main contributors to the generation of 10^14 antibody diversity?

A
  1. Multiple copies of each V region gene segments
    - [Vn x Jn or Vn x Dn x Jn]
  2. Heavy x light chain recombination
    - [Vk x Jk] + [Vl x Jl] x [VH x DH x J]  ~3.3 x 10^6
  3. Recombination is imprecise = junctional diversity(3x10^7)
  4. Somatic mutation of V regions following antigen activation
    - point mutations
    - base changes tend to be clustered in CDRs
    - These are both antigen-dependent
52
Q

what antibody divesity processes are antigen independent?

A
  • multiple copes of each V region gene segments
  • somatic recombination
  • junctional diverity

all of these occur in the bone marrow, so are antigen-independent

53
Q

what antibody diversity process is antigen-dependent?

A

somatic hypermutation occurs in secondary lymphoid tissue when B cells encounter antigen
- so antigen-dependent

54
Q

what is the process of antigen-independent immunoglobulin gene expression?

A

Antigen-independent occurs in bone marrow:
- Heavy chain genes rearrange first (DJ join, then VDJ joining)
- IgM is expressed first
- TdT adds up to 12 nucleotides to VDJ joins - Produces IgM heavy chain
- Light chain gene rearrangement (VJ) to enable expression of IgM at the cell surface
- B cells which recognise the body’s own molecules are deleted
- B cells enter secondary lymphoid tissue where they encounter antigen
- Naïve B cells express membrane IgM or IgM+IgD

55
Q

what is the process of somatic hypermutation (antigen-dependent)? what is the mechanism for this?

A

occurs in secondary lymphoid tissue:
-Point mutations introduced in rearranged V regions (1bp per 10^3 bp per cell division (normal = 1bp per 10^10bp))
- Mechanism: Activation-induced cytidine deaminase (AID), an enzyme expressed by B cells in lymphoid tissue responding to antigen
- Mutations can be introduced throughout V regions BUT in mature B cells, mutations appear to be clustered in CDRs (CDR1,CDR2, CDR3)

56
Q

what is the main function of somatic hypermutation?

A

to trigger affinity maturation

57
Q

what is affinity maturation?

A
  • AFFINITY maturation – higher affinity receptors selected as immune response proceeds - “survival of the fittest”
  • Bind more tightly to antigen
  • Only B cells that bind to antigen are stimulated to divide and differentiate – antigen binding determines B cell survival
  • B cells that don’t bind to antigen die
  • Mutations that increase antigen binding enable B cell survival
58
Q

what is class switching?

A

Class switching: IgM –> IgG, IgA etc.
- same recombined V region associates with different C region genes
- antigen specificity retained, different localisation/effector functions induced
- flexible response to pathogens

59
Q

what do somatic hypermutation and class switching both require?

A

T cell help and AID

60
Q

how does class switching occur?

A

Class switching by DNA recombination between switch regions:
-Double-stranded breaks are generated in DNA at conserved nucleotide motifs, called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody-heavy chains
- intervening DNA lost between switch regions
- irreversible
- Switch sequence allows recombination of VDJ gene to recombine with IgG or IgA constant region gene

61
Q

how do hypermutation and class switching differ in mechanism?

A

Both are initiated by AID, but AID acts at switch regions in class switching (G –rich tandem repeated DNA sequences found close to C region genes).

62
Q

what is the mechanism of action of AID?

A

AID is expressed in activated B lymphocytes, only active on ssDNA
- AID deaminates cytidine to form uracil
- Activity triggers DNA repair pathways which lead to mutations

63
Q

how do repair pathways lead to mutations through AID?

A

Repair pathways in B cells are error-prone, leading to different mutational outcomes:
- Mismatch repair, base excision repair: SOMATIC HYPERMUTATION
- Single strand nicks → double strand nicks: common in G-rich tandem repeat switch regions = CLASS SWITCHING

64
Q

what disease is caused by a mutation in AID?

A

AID mutation causes immunodeficiency: Hyper-IgM Syndrome Type 2

Immunology (8 decks)

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