HIS10 Antibody Structure And Function

Question 1

How can B cells recognise so many different antigens

Answer 1

Gene rearrangement

Question 2

Antigen recognition process

Answer 2

1. Immunoglobulins (Ig)

2. T cell antigen receptor (TCR)

Question 3

Immunoglobulin

Answer 3

• Glycoprotein
• present in serum and tissue fluid
• some carried on B cell surface as receptors for specific antigens
• some free in blood / lymph —> Antibodies
• 10^7-10^8 different specificities

Question 4

Structure of Immunoglobulins

Answer 4

• Y-shaped
• 4 polypeptide chains: 2 Heavy + 2 Light (κ + λ chains)
• Joined by Disulphide bond —> make Fab area
• Both heavy and light composed of Constant + Variable regions

1. V regions (VH + VL) (Amino-terminal sequences):
   - Hypervariable region + Framework region
   - amino acid sequences of both Heavy and Light chains **vary greatly between different Ab
   - Each Ab have 2 **identical Antigen binding sites (each via pairing of VH + VL)
   —> ***Antigen recognition
2. C regions (Carboxy-terminal sequences):
   - **Constant between chains
   —> **Effector functions

(Fab + Fc region: Top + bottom part)

Question 5

V regions

Answer 5

• Sequence variability not distributed evenly throughout
• Background: 5-15% variation (0% for C region)

1. Hypervariable regions (HV1-HV3) / Complementarity determining regions (CDR)
   - Maximum variation in **3 areas of VL / VH domains (same positions in IgG of different specificities)
   —> form loops at the edge
   —> when VH pair with VL
   —> **Hypervariable loops from each domain brought together by folding
   —> single ***Hypervariable surface
   —> Antigen binding site at tip of each arm
   —> Antigen recognition

• Hypervariable regions project around a cleft which is capable of containing an ***Epitope of 6-9 amino acids / carbohydrates

(Hypervariable regions:
H chain residues: 31-37, 51-68, 92-103
L chain residues: 24-34, 50-56, 89-97)

2. Framework regions (FR1-FR4)
   - Intervening regions of **lesser variability (i.e. regions between Hypervariable regions)
   —> **β sheets (provide ***structural framework of domain)

Question 6

***Antigen specificity of combined VH, VL regions

Answer 6

1. ***Amino acid sequences of V regions in the antigen binding site (Hypervariable regions)
2. ***3-dimensional shape of antigen binding site

Question 7

Germline theory, Somatic mutation theory

Answer 7

Germline theory:
- separate gene for each antibody
- antibody repertoire is inherited
—> BUT not enough genes in human body for such diversity

Somatic mutation theory:
- limited number of inherited antibody genes undergo mutation
—> generate repertoire during lifetime of individual

Recent discovery:
- antibody repertoire is generated from large but limited number of Ig genes by DNA **rearrangement
—> diversity enhanced by process of **somatic mutation

Question 8

***Immunoglobulin gene structure

Answer 8

2 functionally distinct groups of exons encoding:
- V region
- C region
—> “2 genes, 1 polypeptide chain”

Ig genes encoding H and L chain are on different chromosomes:

• H chain: 14
• κ chain: 2
• λ chain: 22

C region:
- both H and L chain is encoded by a separate exon

V region in H chain is encoded by 3 gene segments:

1. V (variable)
2. D (diversity)
3. J (joining)

V region in L chain (κ + λ chains) is encoded by 2 gene segments:

1. V (variable)
2. J (joining)

簡單而言:
Different chromosome (for H + κ + λ chains)
—> separate genes for V / C region respectively
—> V region of L chain: 2 exons (V + J) (每一個exon都有幾種gene segments e.g. V1, V2, J1, J2 —> different combinations during gene rearrangement)
—> V region of H chain: 3 exons (V + D + J)
—> C region of L/H chain: 1 exon (C)

Question 9

***Ig gene rearrangement process

Answer 9

H chain genes rearranged first (Pro-B cells stage)
—> then L chain genes (Pre-B cells stage)

H chain: D-J —(DNA recombination)—> V-DJ (i.e. VH) —(DNA recombination)—> VDJ-C —(RNA splicing)—> VDJC
L chain: V-J (i.e. VL) —(DNA recombination)—> VJ-C —(RNA splicing)—> VJC

Steps:
1. **Locating V, D, J gene segments
—> by **RAG-1 and ***RAG-2 genes (Recombination-activating genes)
—> Initiate Ig gene recombination

2. Action by **DNA-PK (cutting), **DNA ligase (joining) etc.
   —> DNA cutting and rejoining (Deletional joining)
   —> able to rearrange different gene segments together
   —> forming functional regions
   (—> still contains additional J + D segments between rearranged segment and C genes)
3. Whilst joining DNA ends of gene segments together
   —> not complementary
   —> require base substitution / **adding of nucleotides to single-strand (broken/naked) DNA ends
   —> by **TdT (Terminal deoxynucleotidyl transferase)
   —> different nucleotides added into junctional region
   —> encoding different amino acids
   —> contribute to ***Junctional diversity
4. Primary RNA transcript produced
   —> mRNA produced
   —> C region genes recombined with VJ / VDJ complex by **RNA splicing
   —> Light chain mRNA: VJC; Heavy chain mRNA: VDJC
   —> Heavy chain: **first transcript always μ (i.e. **IgM)
   —> later in immune response **H chain transcript will change and switch to other isotypes
5. Polypeptides produced

Question 10

***How is Antibody diversity generated?

Answer 10

1. ***Numerous Ig gene segments (V region)
   - H chain: 65x variable, 27x diversity, 6x joining
   - L chain: 70x variable, 5x joining (κ) / 4x joining (λ)
2. Random ***recombination of gene segments
   - H chain: 11,000 gene combinations
   - L chain: 350 gene combinations
3. ***Junctional diversity (x 100 Heavy x 100 Light = 10^4 combinations)
4. ***Pairing of H and L chain (same H chain can combine with different L chain)
   - 11,000 x 350 —> 3.5x10^6 different specificities

Overall:
Total potential antibody diversity at V regions»_space; 10^11 combinations

Question 11

Clinical significance of RAG genes

Answer 11

If lack RAG genes
—> Cannot initiate Ig gene recombination
—> NO functional (mature) B / T cell generated

Severe combined immunodeficiency disease (SCID)

• genetic disorders result in impaired B and T cell immunity
• 1-5 per 500,000
• early death due to severe infection

Question 12

Allelic exclusion

Answer 12

Productive Ig gene rearrangement only occurs on one chromosome
—> ensure each B cell only one specificity (e.g. either κ or λ but not both)

Question 13

How antibodies produced by B cells?

Answer 13

1. Recognition
2. Activation (Proliferation + Differentiation)
3. Reaction
   - Effector B cells
   - Memory B cells

Question 14

Antibody isotopes

Answer 14

IgG, IgM, IgD, IgA, IgE

Variation in Constant region of Heavy chain

B cell can switch from producing one isotype (IgM mainly) to other isotypes during Ab-mediated immune response

Clinical significance:
- Defect in CD40/CD40L signaling
—> Ig cannot undergo class-switching
—> Hyper IgM syndrome (rare, inherited immune deficiency disorder)

Question 15

IgG

Answer 15

• Major Ig in normal human serum (80% of total Ig pool)
• single 4-chain molecule (2 ***γ heavy + 2 κ/λ light)
• 4 subclasses (difference in ***γ chain)
• IgG1, IgG2, IgG3, IgG4 (decreasing average serum concentration)
• distributed evenly between intravascular and extravascular
• Major Ab for ***secondary immune response
• all classes ***cross placenta —> confer high degree of passive immunity to newborn
• Maternal IgG confers immunity to neonates during first few months of life

Question 16

IgM

Answer 16

• 10% of Ig pool
• ***monomeric IgM: membrane-bound Ab on B cells
• first Ig class produced in ***primary response to antigenically complex infectious organisms —> “early” Ab
• first Ig synthesised by neonates
• secreted by plasma cells as a ***pentamer (5 monomer units held by S-S bond linking carboxyl-terminal heavy chain domains)
• each pentamer contains additional Fc-linked polypeptide —> **J chain —> allow IgM to bind to receptors on **secretory cells —> transport ***across epithelial lining to external secretions —> accessory role as secretory Ig
• large size —> does not diffuse well —> largely confined ***intravascularly

Question 17

IgA

Answer 17

• 15-20%
• ***monomer
• polymeric forms (dimers / trimers) (with J chain) sometimes seen
• predominantly in external **seromucous secretions (breast milk, saliva, tears, mucus) as **Dimer / Tetramer + **J chain + **Secretory component polypeptide chain (produced by epithelial cells of mucous membrane)
• transported across cells from tissue fluid to outside environment

Question 18

IgD

Answer 18

• <1% of plasma Ig
• present in large amount on B cell membrane
• functions relatively ***unknown, may play a role in antigen-triggered lymphocyte differentiation

Recent research:
Bind to surface receptor on Basophils
—> activate Basophil
—> enhance immune surveillance (Antibacterial information)

Question 19

IgE

Answer 19

• scarce in serum
• found on surface of **Basophils and **Mast cells
• ***Type 1 hypersensitivity reaction (Allergy: hay fever, asthma, hives, anaphylactic shock)
• role in immunity of ***helminthic parasites

Type 1 hypersensitivity reaction:
IgE bind to Fc receptor on Mast cells
—> allergens bind to IgE
—> trigger ***degranulation of Mast cells
—> Histamine and other substances

Question 20

B cells are bad for heart

Answer 20

1. B lymphocytes trigger monocyte mobilisation
   —> macrophages
   —> induce inflammation
   —> impair healing of heart after acute MI
2. Also involved in Atherosclerosis process