Chapter 4 Flashcards
Antibody structure and B cell diversity
1
Q
What are 5 isotypes of immunoglobulins?
A
IgG, IgD, IgA, IgM, IgE
2
Q
What are parts of antibodies and their function?
A
- heavy and light chains,
- differences in heavy-chain C region ⇒ 5 isotypes of immunoglobulins
- 2 isotypes of light chains: kappa (2/3 of antibodies has this type) and lambda
- each antibody has one or the other type
- N- and C-terminus,
- interchain disulphide bonds,
- there are also intrachain bonds (within a chain)
- variable and constant regions (domains), hinge region,
- each variable region consists of 1 variable domain
- V_H domain + V_L domain
- same for constant region of light chain C_L domain
- constant region of heavy chain ⇒ 3 or 4 domains (depending on isotype)
- both domains form a “sandwich” with B sheets held by hydrophobic interaction + disulphide bonds
- strands are held by loops
- loops differ in V domains near N terminus to form different antigen-binding sites
- each variable region consists of 1 variable domain
- Antigen binding site (Fab= fragment antigen binding) and domains involved in effector functions (Fc= fragment crystallisable)
- effector functions = binding to serum proteins and cell-surface receptors
- these regions can be cleaved by proteasome
- cleavage of unstructured regions of heavy chains = hinge regions
- Fabs in different spacial orientations
- cleavage of unstructured regions of heavy chains = hinge regions
- stability of antibodies comes from immunoglobulin domain
- motifs consisting of 100-110 aa residues
3
Q
How is antigen-binding site formed on a molecular level?
A
- V_H and V_L have hypervariable regions (HVs)
- framerwork regions (FRs) are between HVs
- each chain (light and heavy) has 3 HVs on the loops
- forms variety => complementarity-determining regions (CDRs)
4
Q
To which part of antigen does antibody bind?
A
- epitope
- antigen with more than 1 epitope = multivalent
- antigen can have the same or different epitopes
- binding is non-covalent (electrostatic, H, van der Waals, hydrophobic)
5
Q
How are monoclonal antibodies produced?
A
- B cells from mous immunised with antigen are fused with myeloma cells (tumor)
- grown in drug-containing medium (so only hybridomas survive)
- antigen-specific hybridoma selected and cloned
- grown in tissue culture and properties studied
6
Q
What are types of monoclonal antibodies used for treatment?
A
- mouse -> can be rejected by humans (anti-mouse IgG activated)
- chimeric -> variable regions of mice antibodies fused with constant regions of human ab
- humanised -> only CDR loops are from mice
- fully human
7
Q
What is germline configuration?
A
- arrangement of immunoglobulin genes (for both heavy and light chain) in segments along chromosomes
- segments rearranged → gene formed (immature B cell)
- occurs during development from B cell precursor in bone marrow
- once arranged → produced → found on surface of B cell
8
Q
How are genes coding V region assembled?
A
- V regions are encoded by 2 (V_L) or 3 (V_H) gene fragments
- spliced and cut to produce a V-region exon before transcription
- 2 gene segments= variable gene segments (V) and joining gene segments (J)
- heavy-chain has also diversity gene segments (D) in between V and J
- vary in regions encoding CDR1 and CDR2
- only 1 of the 2 light-chain loci (kappa / lambda) → functional light-chain gene
- spliced and cut to produce a V-region exon before transcription
9
Q
What is somatic recombination?
A
- one of the sources of dversity in B cells
- occurs during B cell development
- in light: only V with J
- in heavy: first DJ → VDJ
- single genes expressed however, many possibilities
- directed by recombination signal sequences (RSSs) = sequences between V and J
- RSS contains heptamers (7 nucleotides or nonamers (9)
- between those are spacers (either 12 or 23 nucleotides long)
- spacers of different length are needed for recombination
- for light: 12/23 rule
- for heavy: only 23 spacers so that D segment (surrounded by 12 spacers) can be added
- RSS contains heptamers (7 nucleotides or nonamers (9)
- V(D)J recombinase = enzymatic system for somatic recombination
- recombination-activating genes (RAG1, RAG2) encode proteins RAG-1, RAG-2
- RAG complex = 2 RAG-1 and 2 RAG-2
10
Q
How does RAG complex work?
A
- 2 RAG-1 form a Y shaped molecule (uneven)
- RAG-2 on the branch of each RAG-1
- branches attach to heptamers, base to nonamers
- junction between heptamer and gene segments (ex. V and J) are formed -> 2 strands are formed
- heptamers are next to gene seq.
- heptamers are joined together (form a loop with other segments incl. nonamers) -> VJ gene formed
- coding (VJ) and signal joint formed
11
Q
How and why is junctional diversity generated?
A
- by enzymes repairing DNA break (after RAG has cut)
- after heptamers are cut off DNA hairpins are formed -> RAG complex splits them and forms a single strand (palindromic sequence, so nucleotides are called P nucleotides)
- TdT = terminal deoxynucleotidyl transferase → randomly adds N-nucleotides
- the 2 strands are paired (unpaired nucleotides removed)
- rest filled in -> double strand formed
- increases diversity on CDR3s (heavy and light)
12
Q
How are IgM and IgD expressed simultaneously?
A
- in a strand for transcription: leader peptide - VDJ region - 9 different segments codng for different C regions (determining isotype)
- Cm and Cd are first, after Cd there’s a stop codon
- only these 2 are expressed
- during splicing it is made sure that either M or D antibody is produced
- specificity is the same (same VDJ region)
- allelic exclusion = only one allele of heavy chain and light chain expressed, the other allele “excluded” or silenced
- ensures that each B cell produces antibodies of a single specificity
13
Q
How does immunoglobulin form a receptor at the B cell membrane?
A
- transported to endoplasmic reticulum → light and heavy chain assembled
- attached to the ER membrane
- transported to B cell surface with help of Igα and Igβ
- same for all immunoglobulins
- transmembrane proteins
- tails conduct signal into the cell after an antigen binds to the receptor
- complex forms a B cell receptor (BCR)
14
Q
What is the difference between membrane-bound and secreted antibodies?
A
- at the C-terminus
- membrane-bound have a hydrophobic part to bind to membrane
- secreted has a hydrophilic segment
- effect achieved by RNA splicing
- hydrophilic segment is more common → 1 exon, while hydrophobic requires joining of 2 exons
15
Q
What is somatic hypermutation and how does it occur?
A
- after B cell activation
- point mutations along rearranged genes coding V region
- high rate
- dependent on enzyme activation-induced cytidine deaminase (AID)
- made by B cell proliferation
- C → U in a ssDNA
- during trasncription (when strands are separated)
- DNA repair enzymes change U to any random base (to rectify the issue)
- substitutions usually in the CDR loops of heavy and light chain → change in antigen binding sites
- may increase affinity of binding
