Ab diversity
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Molecular Basis of Antibody Diversity
-Dr. Nilesh Chandra
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Objectives
• Antibody: structure and properties
• Genetic organisation
• Genetic rearrangement
• Antibody diversity
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Antibody
• Glycoprotein.• Produced by plasma cells.• Recognize & bind antigens.• Lead to:– Phagocytosis– Complement activation– Antibody dependent cell cytotoxicity (ADCC)
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Antibody- structure
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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
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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.
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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.
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Light Chain genes
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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.
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Heavy Chain genes
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Gene rearrangement
• At DNA level• At RNA level
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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.
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Gene rearrangement
• At DNA level• At RNA level
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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.
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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.
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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.
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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.
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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.
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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
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Combinatorial joining of variable region genes
• Random rearrangement of genes from various segments.
• Vast variety of combinations are possible due to this random joining.
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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.
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Junctional flexibility
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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.
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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.
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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.
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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.
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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.
+
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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.
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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
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Summary
• Antibodies and their functions.
• Immunoglobulin gene families (H & L).
• Gene rearrangement (H & L).
• Mechanisms of Antibody diversity.
• Clinical applications.
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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.
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