Antibody structure and the generation of b cell

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  • Antibodies are the secreted form of proteins known more generally as immunoglobulins (Ig). Before it has encountered antigen, a mature B cell expresses immunoglobulin in a membrane-bound form that serves as the B cell’s receptor for antigen. When antigen binds to this receptor, the B cell is stimulated to proliferate and to differentiate into plasma cells, which secrete antibodies of the same specificity as that of the membrane-bound immunoglobulin.
  • Hinge= visagra
  • Antibodies are glycoproteins that are built from a basic unit of four polypeptide chains. This unit consists of two identical heavy chains (H chains) and two identical, smaller, light chains (L chains). Each arm of the Y is made up of a complete light chain paired with the amino-terminal part of a H-chain, covalently linked by a disulfide bond. The two H-chains are linked to each other by disulfide bonds.
  • Variable region - The polypeptide chains of different antibodies vary greatly in amino acid sequence, and the sequence differences are concentrated in the amino-terminal region of each type of chain. This variability is the reason for the great diversity of antigen-binding specificities among antibodies because the paired V regions of a heavy and a light chain form the antigen binding site. Constant region – the remaining parts of the light chain and the heavy chain have much more limited variation in amino-acid sequence between different antibodies.
  • Digestion with the plant protease papain produces three fragment, corresponding to the two arms and the stem. The fragments corresponding to the arms are called Fab (fragment antigen binding) because they bind antigen. The fragment corresponding to the stem is called Fc (fragment crystallizable) because its was seen to crystallize in the first experiments of this sort. Digestion with the gut protease pepsin produces a different fragment, F(ab’) 2 , in which the two arms remain linked by disulfide bonds between the heavy chains.
  • Differences in the heavy chain C regions define five main isotypes or classes of immunoglobulin, which have different functions in the immune response. (IgA, IgD, IgE, IgG, IgM) The light chain has only two isotypes or classes, which are termed kappa and lambda. No functional difference has been found between antibodies carrying kappa chains and those carrying lambda chains.
  • These domains are important because antibodies function in extracellular environments in the presence of infection, where they can encounter variations in pH, salt concentration, proteolytic enzymes, and other potentially destabilizing factors.
  • The heavy and light chains of an immunoglobulin molecule are made from a series of similar protein domains. The V region at the amino terminal end of each heavy chain is composed of a single variable domain (V domain): VH in the heavy chain and VL in the light chain. VH and VL domain together form an antigen binding site. The other domains have little or no sequence diversity within particular isotype and are termed the constant domains (C domains). The constant region of a light chain is composed of a single CL domain, whereas the constant region of a heavy chain is composed of three or four C domains (CH), depending on the isotype.
  • Small differences in shape and chemical properties of the binding site can give several antibodies specificity for the same epitope, but they bind to it with different binding strengths or affinities.
  • For an immunoglobulin gene to be expressed, individual gene segments must be rearranged to assemble a functional gene, a process that occurs only in developing B cells. Immunoglobulin-gene rearrangements occur during the development of B cells from B-cell precursors in the bone marrow. When gene rearrangements are complete, heavy and light chains can be produced and membrane-bound immunoglobulin appears at the B-cell surface. The B cell can now recognize and respond to an antigen through this receptor. Much of the diversity within the mature antibody repertoire is generated during this process of gene rearrangement.
  • In human, the immunoglobulin genes are found at three chromosomal locations: The heavy chain locus – chromosome 14 The к light chain locus – chromosome 2 The λ light chain locus – chromosome 22 Different gene segments encode the leader peptide (L), the V region (V), and the constant region (C) of the heavy and light chains. Gene segments encoding C regions are commonly called C genes. Within the heavy chain locus are C genes for all the different heavy-chain isotypes. The two types of gene segment that encode the light chain V region are called V and J gene segments. The heavy chain locus includes an additional set of D gene segments that lies between the arrays of V and J genes segments.
  • During the development of B cells the arrays of V, D, and J segments are cut and spliced by DNA recombination. This process is called somatic recombination because it occurs in cells of the soma (no germ cells). A single gene segment of each type is brought together to form a DNA sequence encoding the V region of an immunoglobulin chain. For light chains, a single recombination occurs between a V and J segments., whereas for heavy chains, two recombinations are needed, the first to join a D and a J segment, and the second to join the combined DJ segment to V segment. In each case, the particular V, D, and J gene segments that are joined together are selected at random. Because of the multiple gene segments of each type, numerous different combinations of V, D, and J gene segments are possible. Thus, the gene rearrangement process generates many different V-region sequences in the B-cell population. This is one of the factors contributing to the diversity of immunoglobulin V regions.
  • Rearrangement of the V, D, and J segments of the heavy-chain locus brings the gene’s promoter and enhancer into a closer position that enables transcription to proceed. The resulting mRNA is then spliced and translated to give a heavy chain protein. The μ and δ heavy chains genes are the first to be transcribed, so the first immunoglobulins that a B cell expresses on its surface are IgM and IgD. There are the only immunoglobulin isotypes that can be produced simultaneously by a B cell. They are also the only isotypes that B cells produce before they encounter antigen (naïve cells).
  • In each C gene, separate exons encode each of the domains, as is shown for the μ and δ genes in this picture. Transcription starts upstream (RIO ARRIBA) of the exons encoding the leader peptide and the V region, continues through the μ and δ C genes and terminates downstream of the δ gene, before the δ 3 C gene. This long primary RNA transcript is then spliced and processed in two different ways: one that yields mRNA for the μ heavy chain (left panel) and one that yields mRNA for the δ heavy chain (right panel). In making μ chain mRNA from the primary transcript, the entire δ gene is removed as well as the introns from the μ gene. Conversely, in making δ chain mRNA the entire μ gene is removed as well as the introns from the δ gene.
  • This ensure that each B cell produces monoclonal imunoglobulin of a single antigen specificity.
  • Like all proteins destined for the cell surface, immunoglobulin chains enter the endoplasmic reticulum as soon as they are synthesized. There they associated with each other to form immunoglobulin molecules attached to the endoplasmic reticulum membrane. By themselves, these immunglobulin molecules cannot be transported to the cell surface. For that to happen they must associate with two additional transmembrane proteins called Ig α and Ig β . These proteins are invariant in sequence, unlike the immunoglobulins, and travel to the B-cell surface with the immunoglobulins. At the cell surface the complex of immnoglobulin with Ig α and Ig β forms the B-cell receptor for antigen. Ig α and Ig β have long cytolasmic tails that can interact with intracellular signaling proteins.
  • Gene rearrangement in an immature B cell leads to the expression of functional heavy and light chains and to the production of membrane-bound IgM and IgD on the mature B cell. After the encounter with antigen, these isotypes are produced as secreted antibodies. IgM antibodies are produced in large amounts and are important in protective immunity; whereas IgD antibodies are produced only in small amounts and have no known effector function.
  • The surface and secreted forms of an immunoglobulin are derived from the same heavy chain gene by alternative RNA processing. Each heavy chain C gene has two exons (membrane-coding (MS)) encoding the transmembrane region and cytoplasmic tail of the surface form of that isotype, and a secretion-coding (SC) sequence encoding the carboxy terminus of the secreted form. The events that dictate whether a heavy chain RNA will result in a secreted or transmembrane immunoglobulin occur during processing of the initial transcript and are shown here for IgM. Each heavy chain C gene has two potential polyadenylation sites (shown as pAμs and pAμm). In the left panel, the transcript is cut and polyadenylated at the second site (pAμm). Splicing between a site located between the fourth C exon and the SC sequence, an d a second site at the 5’end of the MC exons, removes the SC sequence and joins the MC exons for the fourth C exon. This generates the transmembrane for of the heavy chain. In the right panel, the primary transcript is cleaved and polyadenylated at the first site (pAμs), eliminating the MC exons and giving rise to the secreted form of the heavy chain. AAA designates the poly(A) tail.
  • This almost randomly introduces single-nucleotide substitutions (point mutations) at a high rate throughout the rearranged V regions of heavy and light chain genes.
  • Isotype switching involves recombination between specific switch regions. Repetitive DNA sequences are found to the 5’ side of each of the heavy-chain C genes, with the exception of the δ gene. Switching occurs by recombination between these switch regions (S), with deletion of the intervening DNA. The initial switching event takes place from the μ switch region; switching from other isotypes can take place subsequently.
  • In humans and other mammals the five classes of immunoglobulin are IgA, IgD, IgE, IgG and IgM. IgA- IgA1 and IgA2 Subclasses of IgG – IgG1, IgG2, IgG3 and IgG4, which are numbered according to their relative abundance in plasma, IgG1 being the most abundant.
  • On initiation of an immune response, most of the antibodies that bind the antigen will be of low affinity and the multiple antigen-binding sites of IgM are needed if sufficient antibody is to bind sufficiently strongly to a microorganism to be of any use. When bound to antigen, sites exposed in the constant region of IgM initiate reactions with complement, which can kill microorganisms directly or facilitate their phagocytosis. Synthesis of IgM then gives way to synthesis of IgG.
  • Once they have bound an antigen, the IgG1 and IgG3 subclasses can directly recruit phagocytic cells to ingest the antigen:antibody complex, as well as activating the complement system. During pregnancy, IgG can be transferred across the placenta, providing the fetus with protective antibodies from the mother in advance of possible infection.
  • IgE bound tightly to mast cells triggers strong inflammatory reactions in the presence of its antigens, and is thought to be involved in the expulsion of worms and other parasites. Binding of antigen to receptor-bound IgE induces mast cells to release stored histamine and other activators, which recruit cells and molecules of the immune system to local sites of trauma, causing inflammation.
  • * Example IgA – antibodies bind to a bacterial toxin and neutralize its toxic activity by preventing the toxin from interacting with its receptor on human cells. The complex of toxin and antibodies binds to macrophage receptors through the antibody’s constant region. The macrophage ingests and degrades the complex. Example IgG – the opsonization of a bacterium by coating with antibody. When the bacterium is coated with IgG molecules, their constant regions point outward (HACIA AFUERA) and can bind to the receptor on a macrophage, which then ingests and degrades the bacterium. Example IgM – Opsonization of a bacterium by a combination of antibody and complement. The bacterium is first coated with IgG molecules, which activate complement cleavage. Fragments of complement on the bacterial surface provide ligands for the complement receptor of macrophages. The combined interaction of macrophage receptors for complement and for the constant region of IgG makes for efficient phagocytosis.
  • Antisera – the fluid component of coagulated blood from an immune individual that contains antibodies against an antigen. An antiserum contains a heterogeneous collection of antibodies that bind the antigen. The specificity and quality of such antisera is highly dependent upon the purity of the immunizing antigen preparation because antibodies will be made against all the foreign components it contains.
  • A more modern method for making antibodies does not require a purified antigen. Lymphocytes from a mouse immunized with the antigen are fused with myeloma cells. The cells are then grow in the presence of drugs that kill myeloma cells but permit the growth of hybridoma cells. Individual cultures of hybridomas are tested to determine if they make the desired antibody. The cells are then cloned to produce a homogeneous culture of cells making a monoclonal antibody. Myelomas are tumors plasma cells; those used to make hybridomas were selected not to express heavy and light chains. Thus, hybridomas only express the antibody made by the B cell fusion partner. Hybridoma – hybrid cell lines that make monoclonal antibodies of defined specificity. Monoclonal antibodies – antibodies produced by a single clone of B lymphocytes and that are therefore identical in structure and antigen specificity.
  • Flow cytometry is used to analyze the cell populations in peripheral blood and assess for perturbations caused by disease. The flow cytometer allows individual cells to be identified by their cell-surface molecules. Human cells are labeled with mouse monoclonal antibodies specific for human cell-surface proteins, antibodies of different specificity being tagged with fluorescent dyes of different color.
  • Antibody structure and the generation of b cell

    1. 1. Antibody Structure and the Generation of B - Cell Diversity Dr. Glorivee Rosario Pérez BIOL 4056Parham P. (2009). The Immune System. Third Edition. Garland Publishing, New York.
    2. 2. Introduction Antibodies  Proteins  Produced by the B lymphocytes in response to infection  They circulate as a major component of the plasma in blood and lymph  Their function is to bind to pathogenic microorganisms and their toxins (antigens)  Are specific
    3. 3. Introduction cont.
    4. 4. Figure 3-1
    5. 5. Introduction cont. Antibody production is the single effector function of the B lymphocytes of the immune system.
    6. 6. The structural basisof antibody diversity
    7. 7. Antibodies : Variable and Constant N-terminalregion C-terminal
    8. 8. Antibodies : Variable and Constantregion cont.
    9. 9. Antigen-binding site Hypervariable regions (HV regions)  Small regions of high amino-acid sequence diversity within the variable regions of immunoglobulin and T- cell receptor.  They correspond to the complementarity-determining regions. Complementarity-determining regions (CDRs)
    10. 10. Antibodies : Variable and Constantregion cont.
    11. 11. Antibodies : Variable and Constantregion cont.
    12. 12. Immunoglobulin chains Immunoglobulin domain  Components of protein structure consisting of about 100 amino acids that fold into a sandwich of two β sheets held together by a disulfide bond.  Immunoglobulin heavy and light chain are made up of a series of immunoglobulin domains.
    13. 13. Immunoglobulin chains cont.
    14. 14. Antigen-binding site Hypervariable regions (HV regions)  Small regions of high amino-acid sequence diversity within the variable regions of immunoglobulin and T- cell receptor.  They correspond to the complementarity- determining regions.
    15. 15. Antigen-binding site cont. Complementarity-determining regions (CDRs)  The localized regions of immunoglobulin and T-cell receptor chains that determine the antigenic specificity and bind to the antigen.  The CDRs are the most variable parts of the variable domains and are also called hypervariable regions.
    16. 16. Antigen-binding sites Antigenic determinant (epitope)  The part of the antigen to which an antibody binds.  These structures are usually either carbohydrate or protein, or both, because the surface molecules of pathogens are commonly glycoproteins, polysaccharides, glycolipids, and peptidoglycans.
    17. 17. Figure 3-8
    18. 18. Antigen-binding sites cont. Multivalent antigen – any antigen that contains more than one epitope, or more than one copy of the same epitope.
    19. 19. Antigen-binding sites cont. The binding of antigens to antibodies is based on noncovalent forces:
    20. 20. Generation ofimmunoglobulin diversityin B cells beforeencounter with antigen
    21. 21. Gene segments Multiple short DNA sequences in the immunoglobulin and T-cell receptor genes. These can be rearranged in many different combinations to produce the vast diversity of immunoglobulin or T-cell receptor polypeptide chain.
    22. 22. The organization of the humanimmunoglobulin heavy- and light-chainloci
    23. 23. Figure 4-3
    24. 24. Gene segments (V) Variable (V) gene segments  DNA sequence in the immunoglobulin or T-cell receptor genes that encodes the first 95 or so amino acids of the V domain.  There are multiple different V gene segments in the germINAL genome.  To produce a complete exon encoding a V domain, one V gene segment must be rearranged to join up with a J or a rearranged DJ gene segment.
    25. 25. Gene segments (J) Joining (J) gene segments  One of the types of gene segment in immunoglobulin and T-cell receptor genes that is rearranged to make functional variable- region exons.
    26. 26. Gene segments (D) Diversity (D) gene segments  Short DNA sequence present in immunoglobulin heavy chain loci and in T-cell receptor β- and δ-chain loci.  In the rearranged functional genes at these loci, a D region connects the V and J region.
    27. 27. Mechanisms contributing to thediversity in V-region1. Random combination of different V and J segments in light chains genes and of different V, D, and J segments in rearranged heavy chain genes.2. Introduction of additional nucleotides at the connections between gene segments during the process of recombination.3. Diversity in the antigen-binding sites of antibodies is the association of heavy and light chains in different combinations.
    28. 28. 1. Random recombination of gene segmentsproduces diversity in the antigen-bindingsites of immunoglobulins
    29. 29. Figure 4-2
    30. 30. Figure 4-13
    31. 31. Naïve B cells A mature B cell that has left the bone marrow but has not yet encountered its specific antigen. IgM and IgD
    32. 32. Expression of IgM and IgD
    33. 33. Allelic exclusion In a developing B cell, the process of immunoglobulin-gene rearrangement is controlled so that only one heavy chain and one light chain are finally expressed.
    34. 34. Membrane-bound immunoglobulins
    35. 35. Diversification ofantibodies after B cellsencounter antigen
    36. 36. Secreted antibodies Mature cell (naïve cell) IgM (membrane-bound) IgDantigen antigen ↑↑↑IgM (secreted antibodies) ↑IgD
    37. 37. The surface and secreted forms ofan immunoglobulin
    38. 38. Somatic hypermutation Mutation that occur at high frequency in the rearranged variable region DNA of immunoglobulin genes in activated B cells, resulting in the production of variant antibodies, some of which have a higher affinity for the antigen.
    39. 39. Isotype switching
    40. 40. The physical properties of thehuman immunoglobulin isotypes
    41. 41. Figure 4-18
    42. 42. Figure 4-23
    43. 43. Antibody function : IgM Is the first antibody produced in an immune response. It is made principally by plasma cells resident in:  Lymph nodes  Spleen (BASO)  Bone marrow Circulates in blood and lymph.
    44. 44. Antibody function : IgG Is the most abundant antibody in the internal body fluids, including blood and lymph. It is made in the:  Lymph nodes  Spleen  Bone marrow Is smaller and more flexible than IgM, properties that give it easier access to antigens in the extracellular spaces of damaged and infected tissues.
    45. 45. Antibody function : IgA Is made by plasma cells in:  Lymph nodes  Spleen(HIGADO)  Bone marrow Is secreted into the bloodstream. Dimeric IgA is made in the lymphoid tissues fundamental mucosal surfaces. Is the antibody that is secreted into the lumen of the gut. Principal antibody of milk, saliva, sweat, and tears.
    46. 46. Antibody function : IgE Is highly specialized towards the activation of mast cells, which are present in epithelial tissues. The major impact of IgE is in the allergies that result when it is produced against antigens.
    47. 47. Mechanisms by which antibodiescombat infection
    48. 48. Functions and properties of immunoglobulin
    49. 49. Applications:Antigen-Antibody
    50. 50. Figure A-1
    51. 51. Figure A-5
    52. 52. Figure A-6
    53. 53. Figure A-7
    54. 54. Figure A-8
    55. 55. Figure A-9
    56. 56. Figure A-10
    57. 57. Figure A-13
    58. 58. Monoclonal antibodies The traditional method for making antibodies of desired specificity is to immunize animals with the appropriate antigen and then prepare antisuero from their blood. antisera
    59. 59. Monoclonal antibodies cont.
    60. 60. Monoclonal antibodies cont.
    61. 61. Figure A-15
    62. 62. Radioimmunoassay (RIA) FIGURA 18.37
    63. 63. Radioimmunoassay (RIA) cont.Figure A-18
    64. 64. Figure A-20
    65. 65. Figure A-22
    66. 66. Figure A-23
    67. 67. Figure A-26
    68. 68. Figure A-29
    69. 69. Figure A-40
    70. 70. Figure A-41
    71. 71. ImmunofluorescenceFigure A-16
    72. 72. Immunofluorescence cont.Figure A-17
    73. 73. Immunofluorescence cont. FIGURA 18.35
    74. 74. Immunofluorescence (ELISA) cont. FIGURA 18.38
    75. 75. Immunofluorescence (Western Blot) cont. FIGURA 18.39
    76. 76. Flow cytometry
    77. 77. Flow cytometry cont.Figure A-25 part 2 of 2

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