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ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY. WHAT ARE ANTIBODIES?. Antigen specific proteins produced by plasma cells Belong to immunoglobulin superfamily Located in blood and extravascular tissues, secretions and excretions

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ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

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  1. ANTIBODY STRUCTURE AND THE GENERATION OF B-CELL DIVERSITY

  2. WHAT ARE ANTIBODIES? • Antigen specific proteins produced by plasma cells • Belong to immunoglobulin superfamily • Located in blood and extravascular tissues, secretions and excretions • Bind pathogenic microorganism and their toxins in extracellular compartments • Secreted form of immunoglobulins

  3. WHAT ARE IMMUNOGLOBULINS? • Antigen specific proteins produced by B lymphocytes • Belong to immunoglobulin superfamily • Bound to surface of B lymphocytes • Function as binding (receptor) sites for specific antigens • Antigen receptor sites on mature B lymphocytes • IgM • IgD • Membrane-bound form of immunoglobulins

  4. WHAT IS THE IMMUNOGLOBULIN SUPERFAMILY • Proteins with structural feature first defined in immunoglobulins • Characteristic structural feature • Sequence of Domains providing stable conformation • Domain • Polypeptide (100 to 110 amino acids) chain folded into sandwich (2 slices of bread) held together by disulfide bond • IG superfamily members • Antibodies, B cell receptors, T cell receptors, MHC molecules and others

  5. STRUCTURE OF ANTIBODIES • Antibodies are glycoproteins composed of • Polypeptide chains and carbohydrate • Monomeric structure • Polypeptide chains • 2 identical heavy chains • 2 identical light chains • Polypeptide chains joined by disulfide bonds • Carbohydrate

  6. STRUCTURE OF ANTIBODIES • Polypeptide chains have variable and constant regions • Variable • N (amino)-terminal of polypeptide chain • Antigen binding site • Constant • C (carboxyl)-terminal of polypeptide chain • Binding sites for cell surface receptors and complement • Structure represented by the letter “Y” • Y shaped molecule cleaved by protease papain • Fragment antigen binding (Fab) • Fragment crystallizable (Fc)

  7. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS • Classes based on constant region of heavy chains • Immunoglobulin A (IgA) • Immunoglobulin D (IgD) • Immunoglobulin E (IgE) • Immunoglobulin G (IgG) • Immunoglobulin M (IgM) • Differentiation of heavy chains • Length of C region, location of disulfide bonds, hinge region, distribution of carbohydrate • Classes have different effector functions

  8. CLASSES (ISOTYPES) OF IMMUNOGLOBULINS • Additional classification based on light chains • Kappa • Lambda • Each IG has either kappa or lambda, not both • IgG kappa • IgG lambda • No functional differences between light chains

  9. IT’S GREEK TO ME Heavy chains, light chains and other molecules of the immune system identified using letters of the Greek alphabet

  10. THREE DIMENSIONAL STRUCTURE OF ANTIBODIES • Antibodies function in setting of infectious process • Proteolytic enzymes, salt and pH differences • Antibodies remain stable based on • Sequence of domains • Single domain consists of • 100 – 110 amino acids folded into compact and stable conformation • Domains • Variable (V) • Single V domain in H and L chains • Constant (C) • Single C domain in L chains • Three to four (C) domains in H chains

  11. ANTIGEN BINDING SITES OF IMMUNOGLOBULINS • Antigen binding sites formed from hypervariable regions • Heavy chain V domain • Light chain V domain • Hypervariable regions of V domains • Amino acid sequence differences concentrated • Flanked by less variable framework regions • Three hypervariable regions in each V domain • Hypervariable regions also called • Complementarity-determining regions (CDR)

  12. ANTIGEN BINDING SITES OF IMMUNOGLOBULINS • Antigen binding sites vary with size and shape of antigen • Part of antigen to which antibody binds • Antigenic determinant (Epitope) • Antigen-Antibody binding based on non-covalent forces • Hydrogen bonds • Affinity • Strength of binding of one molecule to another by a single binding site • Avidity • Overall strength of binding between two molecules

  13. ANTIBODIES AS DIAGNOSTIC AND THERAPEUTICS AGENTS • Based on specificity and affinity of antibodies • Both applications require large quantities of identical antibodies • Monoclonal antibodies • Monoclonal antibodies are produced using hybridoma cell line • Hybridoma cell line • Derived from single antibody producing cell fused with myeloma cell (neoplastic plasma cell)

  14. IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN • Immune system capable of producing a limitless number of different immunoglobulins/antibodies • Mechanism • Genes for IG organized differently • Genes exist as nonfunctional segments • Variable (V), Joining (J), Diversity (D), Constant (C) • Genes are inherited in this form • Germline form (germline configuration)

  15. IMMUNOGLOBULIN DIVERSITY IN B-CELLS BEFORE ENCOUNTER WITH ANTIGEN • Expression • Gene segments must be rearranged into functional gene • Gene Rearrangement • Takes place during development of B-cells • Mechanism of somatic recombination • Genes for IG located at 3 chromosomal locations • Heavy chain locus on chromosome 14 • Kappa light chain locus on chromosome 2 • Lambda light chain locus on chromosome 22

  16. GERMLINE ORGANIZATION OF HUMAN IG HEAVY CHAIN AND LIGHT CHAIN LOCI • Lambda light chain locus • Gene segments • 30 (V), 4 (J) and 4 (C) • Kappa light chain locus • Gene segments • 40 (V), 5 (J) and 1 (C) • Heavy chain locus • Gene segments • 65 (V), 27 (D), 6 (J) and 9 (C)

  17. CONSTRUCTION OF LIGHT CHAIN AND HEAVY CHAIN VARIABLE REGIONS • Light chain • Constructed from 2 segments • 1 (V) segment • 1 (J) segment • Heavy chain • Constructed from 3 segments • 1 (V) segment • 1 (D) segment • 1 (J) segment

  18. SOMATIC RECOMBINATION • Performed by enzymes with cut and rejoin DNA • Directed by • Recombination signal sequences (RSS) • Recombination signal sequences • Recognition sites for enzymes • Recombination occurs between different types • 9 / 12 / 7 • 9 / 23 / 7 • Mechanism follows the 12/23 rule • Ensures segments joined in correct order

  19. MECHANISMS OF GENETIC DIVERSITY IN V-REGION OF IMMUNOGLOBULINS • Random combination of • V and J segments in light chain genes • V, D and J segments in heavy chain genes • Addition of P (palindromic) and N (non-templated) nucleotides at junctions of gene segments during recombination • Junctional diversity • Association of H and L chains in different combinations

  20. CONSTRUCTION OF B-CELL SURFACE IMMUNOGLOBULINS • Following rearrangement of VH gene segments, two CH loci are transcribed • IgM • IgD • M and D constant segments • Located nearest variable segments • M and D transcript processed by • Cleavage, polyadenylation and splicing • IgM and IgD enter endoplasmic reticulum

  21. SURFACE IMMUNOGLOBULINS ASSOCIATED WITH PROTEINS TO COMPLETE ANTIGEN RECEPTOR • In ER, IgM and IgD associated with transmembrane proteins • Ig-alpha • Ig-beta • Transmembrane proteins • Transport M and D to B cell surface • Communication of antigen binding to inside of B cell • Tails interact with intracellular signaling molecules • Complex of IgM and IgD with Ig-alpha and Ig-beta forms • B-cell receptor

  22. DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN • Mature, naïve B cell has membrane bound IgM and IgD antigen receptors • Binding of antigen initiates proliferation and differentiation of B-cells into plasma cells • During differentiation, B cells switch from making immunoglobulin to antibody M and D isotypes • IgM • Produced in large amounts • Provides protective immunity • IgD • Produced in small amounts • No known function

  23. MECHANISM OF SWITCHING FROM IMMUNOGLOBULIN TO ANTIBODY • Surface and secreted forms derived from same heavy chain gene by alternative RNA processing • Each heavy chain C gene has • Membrane coding (MC) region • Secretion coding (SC) region • Mechanism involves a switch in cleavage, polyadenylation and splicing • From pAm region to pAs region

  24. DIVERSIFICATION OF ANTIBODIES AFTER B-CELLS ENCOUNTER ANTIGEN • Following antigen activation of B-cells, additional diversification occurs in V domain by • Somatic hypermutation • Somatic hypermutation • Introduction of random single nucleotide substitutions (point mutations) throughout V regions of H and L chains • Mechanism poorly understood • More common in hypervariable regions (CDRs)

  25. OUTCOME OF SOMATIC HYPERMUTATION • Gives rise to some antibodies with higher • Affinity for antigen • Affinity • Strength of binding of one molecule to another by a single binding site • Higher affinity antibodies are produced as immune response proceeds • Affinity maturation

  26. THE PRIMARY HUMORAL IMMUNE RESPONSE • Immune response initially produces IgM antibodies then switches to IgG antibodies • Question • Why switch from IgM to IgG? • Answer • Limited effector mechanisms for IgM • Range of effector mechanisms for IgG • Mechanism • Isotype or class switching

  27. ISOTYPE OR CLASS SWITCHING • Process by which B cell changes class of IG produced while preserving antigenic specificity • Involves somatic recombination which attaches different heavy chain constant region to variable region • Occurs only during active immune response • Mechanisms involves recombination between • Switch sequences (regions)

  28. CLASSES, SUBCLASSES AND PHYSICAL PROPERTIES OF IMMUNOGLOBULINS Subclasses are numbered according to plasma concentration

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