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Molecular Basis of Antibody
Diversity
-Dr. Nilesh Chandra
Objectives
• Antibody: structure and properties
• Genetic organisation
• Genetic rearrangement
• Antibody diversity
Antibody
• Glycoprotein.
• Produced by plasma cells.
• Recognize & bind antigens.
• Lead to:
– Phagocytosis
– Complement a...
Antibody- structure
Functions of Antibodies
• B cell Ag receptor as mAb.
• Neutralization of Ag by sAb.
• Complement activation
• Opsonization...
Immunoglobulin gene families
• Located on different chromosomes for
different chains:
– H-chain gene family on Ch 14.
– Ka...
Light Chain genes
• Encoded by 3 genes:
– V (variable gene)
– J (joining gene)
– C (constant gene)
• V & J together code f...
Light Chain genes
Heavy Chain genes
• Encoded by 4 genes:
– Variable region coded by 3 genes:
• VH (variable)
• JH (joining)
• DH (diversity...
Heavy Chain genes
Gene rearrangement
• At DNA level
• At RNA level
Rearrangement at DNA level
• V,D & J joining in H-chains; V & J joining in L-
chains.
• C region genes remain separated fr...
Gene rearrangement
• At DNA level
• At RNA level
Rearrangement at RNA level
• Takes place during the primary RNA transcript
processing.
• The C-region gene spliced with V-...
Heavy chain gene rearrangement
• Rearranged V region at DNA level conatins the
following sequences from 5’ end to 3’ end:
...
Heavy chain gene rearrangement
• The rearranged H-gene is transcribed.
• Next rearrangement at RNA level takes place.
• Di...
Light chain gene rearrangement
• Occurs after the rearrangement in heavy
chain.
• Similar in pattern.
• No D-genes involve...
Antibody Diversity Mechanism
• Multigene organisations of Ig chain.
• Combinatorial joining of variable region genes.
• Ju...
Multigene organisations of Ig chain
• Large number of variable region genes:
• David Parry
Gene
segment
Number of genes
He...
Combinatorial joining of variable
region genes
• Random rearrangement of genes from various
segments.
• Vast variety of co...
Because of the diversity contributed by junctional flexibility, P- & N-
region nucleotide addition, and somatic mutation, ...
Junctional
flexibility
P-nucleotide addition
• During DNA rearrangement, a hair-pin structure is
formed.
• This hairpin is later cleaved by an en...
N-nucleotide addition
• Addition of new nucleotides at the free 3’ end.
• By enzyme terminal deoxynucleotide
transferase (...
Somatic hypermutation
• V-region genes in B-cell, on antigenic
stimulation, undergo point mutations.
• Mutations result fr...
Combinatorial association of H- and L-
chain
• Specificity of antibody is determined by the V-
region of both the H & L ch...
Because of the diversity contributed by junctional flexibility, P- & N-
region nucleotide addition, and somatic mutation, ...
Multiple myeloma
• Malignant disease of plasma cells.
• Normally, H & L chains are produced in equal
amounts.
• In multipl...
Clinical applications
• Understanding of immunoglobulin structure
and formation has opened up a new world of
possibilities...
Summary
• Antibodies and their functions.
• Immunoglobulin gene families (H & L).
• Gene rearrangement (H & L).
• Mechanis...
References
• Immunology, 5th Edition; Kuby
• Roitt’s Essential Immunology, 10th edition
• Essentials of Immunology; S.K.Gu...
THANK YOU!
Ab diversity
Ab diversity
Ab diversity
Ab diversity
Ab diversity
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Ab diversity

  1. 1. Molecular Basis of Antibody Diversity -Dr. Nilesh Chandra
  2. 2. Objectives • Antibody: structure and properties • Genetic organisation • Genetic rearrangement • Antibody diversity
  3. 3. Antibody • Glycoprotein. • Produced by plasma cells. • Recognize & bind antigens. • Lead to: – Phagocytosis – Complement activation – Antibody dependent cell cytotoxicity (ADCC)
  4. 4. Antibody- structure
  5. 5. Functions of Antibodies • B cell Ag receptor as mAb. • Neutralization of Ag by sAb. • Complement activation • Opsonization • ADCC • Mucosal immunity • Neonatal immunity (by IgG & IgA) • Immediate hypersensitivity by IgE
  6. 6. Immunoglobulin gene families • Located on different chromosomes for different chains: – H-chain gene family on Ch 14. – Kappa light chain gene family on Ch 2. – Lambda light chain gene family on Ch 22. • One family multiple gene segments. • One gene segment multiple genes for same specific region.
  7. 7. Light Chain genes • Encoded by 3 genes: – V (variable gene) – J (joining gene) – C (constant gene) • V & J together code for variable region ( VL). • C gene codes for the constant region ( CL). • A complete L chain formed by splicing of V, J & C genes.
  8. 8. Light Chain genes
  9. 9. Heavy Chain genes • Encoded by 4 genes: – Variable region coded by 3 genes: • VH (variable) • JH (joining) • DH (diversity) –Constant region (CH) encoded by one single gene. –H-chain C-segment has 9 genes.
  10. 10. Heavy Chain genes
  11. 11. Gene rearrangement • At DNA level • At RNA level
  12. 12. Rearrangement at DNA level • V,D & J joining in H-chains; V & J joining in L- chains. • C region genes remain separated from V- region gene by J genes and intron. • A primary RNA transcript is generated. • Only Cμ & Cδ genes are transcribed in naïve B cells. • The other C region genes transcribed during class switching.
  13. 13. Gene rearrangement • At DNA level • At RNA level
  14. 14. Rearrangement at RNA level • Takes place during the primary RNA transcript processing. • The C-region gene spliced with V-region genes to generate complete H & L chain genes. • Plays crucial role in: – Production of membrane bound Ab. – Production of secreted form of Ab. – Production of different classes of Ab.
  15. 15. Heavy chain gene rearrangement • Rearranged V region at DNA level conatins the following sequences from 5’ end to 3’ end: – L-exon of joined VH gene. – VHDHJH –combined exon. – Intron between L & VHDHJH. – Intron 3’ to J gene. – Remaining JH genes, if any, followed by complete set of CH region genes.
  16. 16. Heavy chain gene rearrangement • The rearranged H-gene is transcribed. • Next rearrangement at RNA level takes place. • Differential splicing of the primary RNA transcript leads to generation of mRNA for both μ and δ heavy chains. • After translational, leader peptide is cleaved to produce a fully functional chain.
  17. 17. Light chain gene rearrangement • Occurs after the rearrangement in heavy chain. • Similar in pattern. • No D-genes involved. – Variable region rearrangement in DNA. – Splicing with C-region gene during RNA processing.
  18. 18. Antibody Diversity Mechanism • Multigene organisations of Ig chain. • Combinatorial joining of variable region genes. • Junctional flexibility. • P-nucleotide addition. • N-nucleotide addition. • Somatic hypermutation. • Combinatorial association of H- and L-chain.
  19. 19. Multigene organisations of Ig chain • Large number of variable region genes: • David Parry Gene segment Number of genes Heavy chain Kappa chain Lambda chain V 51 40 30 D 27 0 0 J 6 5 4
  20. 20. Combinatorial joining of variable region genes • Random rearrangement of genes from various segments. • Vast variety of combinations are possible due to this random joining.
  21. 21. Because of the diversity contributed by junctional flexibility, P- & N- region nucleotide addition, and somatic mutation, the actual potential exceeds these estimates by several orders of magnitude. +
  22. 22. Junctional flexibility
  23. 23. P-nucleotide addition • During DNA rearrangement, a hair-pin structure is formed. • This hairpin is later cleaved by an endonuclease. • A short single strand at the end of the coding sequence is left. • The subsequent addition of complementary nucleotides to this strand (P-addition) by repair enzymes generates a palindromic sequence in the coding joint, hence called P-nucleotides. • Variation in the position at which the hairpin is cut thus leads to variation in the sequence of the coding joint.
  24. 24. N-nucleotide addition • Addition of new nucleotides at the free 3’ end. • By enzyme terminal deoxynucleotide transferase (TdT). • Up to 15 nucleotides can be added at the coding joints. • Contribute to antibody diversity.
  25. 25. Somatic hypermutation • V-region genes in B-cell, on antigenic stimulation, undergo point mutations. • Mutations result from nucleotide substitution. • Seen in response to T-cell dependent protein antigens. • Influence the process of affinity maturation.
  26. 26. Combinatorial association of H- and L- chain • Specificity of antibody is determined by the V- region of both the H & L chains. • The possible combinations of the H and L chains are also a source of antibody diversity.
  27. 27. Because of the diversity contributed by junctional flexibility, P- & N- region nucleotide addition, and somatic mutation, the actual potential exceeds these estimates by several orders of magnitude. +
  28. 28. Multiple myeloma • Malignant disease of plasma cells. • Normally, H & L chains are produced in equal amounts. • In multiple myeloma, L-chains are synthesized much in excess than H-chains. • Urine of these patients show increased amounts of L-chain secretion.
  29. 29. Clinical applications • Understanding of immunoglobulin structure and formation has opened up a new world of possibilities: – Monoclonal antibodies – Engineering mice with human immune systems – Generating chimeric and hybrid antibodies for clinical use – Abzymes: antibodies with enzyme capability
  30. 30. Summary • Antibodies and their functions. • Immunoglobulin gene families (H & L). • Gene rearrangement (H & L). • Mechanisms of Antibody diversity. • Clinical applications.
  31. 31. References • Immunology, 5th Edition; Kuby • Roitt’s Essential Immunology, 10th edition • Essentials of Immunology; S.K.Gupta • Diverse functions for DNA and RNA editing in the immune system. Hamilton CE, Papavasiliou FN, Rosenberg BR. RNA Biol. 2010 Mar-Apr;7(2):220- 8. Epub 2010 Mar 29. • B cells from the bench to the clinical practice. Moura R, Agua-Doce A, Weinmann P, Graça L, Fonseca JE. Acta Reumatol Port. 2008 Apr- Jun;33(2):137-54.
  32. 32. THANK YOU!
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