Medical Immunology (Exam II material) Lecture 4

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Medical Immunology (Exam II material) Lecture 4
2015-03-11 23:46:20

Antibody Structure and Generation of B-Cell Diversity Parham Ch. 4
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  1. Ab’s have variable regions, which determine Ag specificity, and constant regions which determine effector function and isotype. What are the five types and there where are their structural difference?
    • IgA, IgD, IgE, IgG, and IgM
    • Their structural differences reside in their constant regions
  2. What is the most abundant antibody in the blood and lymph?
  3. How many polypeptide chains are in an IgG molecule?
    • Composed of 4 polypeptide chains:
    • 2 identical heavy chains
    • 2 identical light chains
  4. What does on arm of an IgG antibody consist of?
    1 light chain + the N-terminal half of the heavy chain
  5. Where is the variable region, what does it consist of, and what is its function?
    • -the variable region is located at the tip of the antibody arm
    • -consists of the N-termini of the heavy and light chain polypeptides
    • -is responsible for for antigen specificity
  6. What does the stem of the IgG antibody consist of and what is its function?
    • -composed of 2 C-terminal halves of the heavy chains
    • -the stem determines effector function and isotope
  7. Where do disulfide bonds form links in the IgG antibody?
    • - between the light and heavy chains in each arm
    • -and between the heavy chains in the stem of the antibody
  8. What enzyme is responsible for digestion of IgG?
  9. How many fragments are yielded with the digestion of IgG antibody, with papain?
    • -2 Fab
    • -2 Fc
  10. What does Fab stand for and where is it located?
    • -Fragment Antigen Binding
    • -located in the arms of the antibody
  11. What doe Fc stand for and where is it located?
    • -Fragment Crystallizable
    • -located in the stem of the antibody
  12. Antibodies are glycosylated.

    A. True
    B. False
    A. TRUE
  13. What and where are the connections allowing for IgG flexibility of the antigen-recognizing arms?
    • -the hinge region
    • -located on the arms (Fab's) connecting to the stem (Fc)
  14. What antibody(s) do NOT have hinge regions?

    Which do?
    -IgM and IgE do NOT have hinge regions

    -IgA, IgD, IgE, and IgG have hinge regions
  15. How many amino acids does an Ig domain have?
    -approximately 100-110 amino acids
  16. How many Ig domains does IgM have? How many amino acids?
    • -Ig 70 domains
    • - approximately 70,000 amino acids
  17. What kind of structure is an Ig domain and what does the secondary structure consist of?
    • -an Ig domain is a protein tertiary structure
    • -its secondary structure consists of beta strands that form two sheets stacked like a sandwich
  18. An Ig domain is compact and very stable. It can withstand diverse extracellular conditions without_______. Antibody protein must be properly_______to have antigen specificity.
    denaturing; folded
  19. How many domains does each arm of an Ig antibody consist of?
    -4 Ig domains; 2 from the light chain, 2 from the heavy chain
  20. Which antibodies have 4 domains that make up their stem?
    -IgA, IgD, and IgG
  21. Which antibodies have 6 domains that make up their stem?
    -IgM and IgE
  22. Ig = Ig domain

    A. True
    B. False
    B. FALSE

    Ig refers to a FULL immunoglobulin composed of many Ig domains
  23. What domains do both light and heavy chains have?
    -Variable domain and constant domain
  24. Each chain has exactly one of which domain? What are the rest?
    • -only one V domain
    • -the are C domains
  25. What is the composition of the light chain in regards to domain?
    - One Variable and one Constant domain per light chain
  26. What is the composition of the heavy chain in regards to domain?
    • -one Variable domain and either 3 or 4 Constant domains
    • -a total of 4 or 5 Ig domains per heavy chain
  27. Which Ig antibodies have 3 Constant domains per heavy chain?
    IgA, IgD, and IgG

    (these isotopes have flexible hinge region connecting arms to stem)
  28. Which Ig antibodies have 4 Constant domains per heavy chain?
    IgM and IgE

    (these antibodies do NOT have a flexible hinge region connecting arms to the stem)
  29. Ig-like domains are NOT common in cell-surface and secreted proteins of the immune system.

    A. True
    B. False
    B. FALSE

    Ig domain is a reoccurring protein motif; Ig-like domains ARE COMMON in cell surface and secreted proteins of the immune system
  30. What regions does the antigen-binding site consist of?
    hypervariable regions of the Variable light and Variable heavy Ig domains.
  31. The hyper variable regions of the Variable Ig domain have very high_______composition diversity between different Ig's.
    -amino acid
  32. As opposed to the secondary structure of an Ig, which consists of beta strands, what do the hyper variable regions consist of?
    -loop regions
  33. What are the hyervariable regions ALSO known as?
    - CDR's or complementary determining regions

    (as they provide binding surface complementary to the epitope of an antigen)
  34. What is the difference between an epitope and an antigen?
    • -an epitope is a molecular feature recognized by a receptor 
    • -an antigen is a molecular entity that CONTAINS an epitope
  35. What is the difference between specificity and affinity?
    • -specificity is when one molecule binds selectively to ONLY one other molecule
    • -Affinity is the STRENGTH of that molecular binding (the stronger the affinity, the stronger the binding and vice versa)
  36. What determines an antibodies effectiveness against a particular pathogen?
    - the specificity and affinity for an antigen for that pathogen.
  37. In thermodynamic terms, how does a high affinity correlate between binding affinity and ΔG'o?
    - high binding affinity gives a large negative ΔG'o
  38. List some examples of pathogen antigens
    glycoproteins, polysaccharides, glycolipids, peptidoglycans
  39. An epitope is also known as an________.
    -antigenic determinant

    (the part of an antigen that antibodies bind)
  40. Can antigens have more than one epitope and if so, can they be the same epitope or different epitopes?

    A. Yes
    B. No
    A. YES

    Antigens can have multiple epitopes and display all of the same or completely different epitopes
  41. What is an antigen with multiple antigens called?
    multivalent antigen
  42. Only a small portion of a virus capsid (~2-10 amino acids) is the actual epitope.

    A. True
    B. False
    A. TRUE

    a virus capsid consists of hundreds to thousands of amino acids but only 2-10 are recognized
  43. What form do antigen-binding sites take to complement epitope of a small molecule?
    -a pocket
  44. What form do antigen-binding sites take to complement epitope of a linear molecule?
    -a groove
  45. What form do antigen-binding sites take to complement epitope of a large molecule?
    -flat extended surface
  46. What type of bond interaction exists between antibodies and antigens?
    • -No chemical bonds exist between these molecules
    • -binding is non-covalent: electrostatic, van der Waals, hydrogen, hydrophobic, aromatic
  47. Monoclonal antibodies are NOT identical.

    A. True
    B. False
    B. False

    monoclonal antibodies are identical
  48. What make a B or T cell a "clone"?
    - when they express identical Ig or TCR's
  49. Why are monoclonal antibodies identical?
    -because they express identical Ig or TCR's due to being produced by a single hybridoma clone
  50. What characteristics do antibodies share, besides their structure and appearance?
    -all have the same specificity and affinity to the same antigen, and the SAME EPITOPE on that antigen.
  51. How would monoclonal antibodies be useful in a lab?
    -monoclonal antibodies can be used as a way to selectively label a given compound due to specific antibody detection for that compound, such as cell surface proteins.
  52. What does "CD" stand for, as in CD4 or CD8?
    -CD stands for cluster differentiation

    -each CD refers to a particular type of cell-surface protein
  53. Approximately, how many CD markers have been identified and how?
    • -there are >300 CD markers
    • - they were discovered using monoclonal antibodies
  54. Mouse derived monoclonal antibodies cannot be used in humans. What part of this antibody will generate a human immune response?

    How can we resolve this issue?
    -the constant regions, composed of both heavy and light domains

    -a "chimeric" monoclonal antibody can be produced, converting the constant region to a human version
  55. What does "mab" stand for in rituximab? What is this medication used to treat?
    -mab stands for monoclonal Ab

    -used to treat certain types of NON-Hodgkins Lymphoma
  56. Humanized monoclonal antibodies are produced by humanizing everything BUT...
    CDR (complementary determining region) loops
  57. What is the difference between affinity and avidity?
    -affinity is the strength of a SINGLE binding site with an antigen

    -avidity is the strength of MULTIPLE antigen binding sites to MULTIPLE antigen
  58. IgMs can achieve high avidity for multivalent antigens, why is this?
    -IgM's consist of 5 Ig antibodies attached by a J chain. Each Ig antibody contains 2 antigen binding sites. 5 x 2 = 10 antibody binding sites per pentamer (IgM)
  59. What is the first antibody produced in an immune response?
  60. What are the mechanisms by which antibodies are used to fight infection?
    • -Neutralization
    • -Opsonization
    • -Mast cell activation
    • -Isotype switching
  61. What is isotope switching and where do you see it occurring during a mounted immune response?
    -isotype switching is a class switch recombination in an antibody.

    • They occur during:
    • -Blood infections: IgM-->IgG
    • -Gut infections: IgM-->IgA
    • -Parasite infections: IgM-->IgE
  62. What is mast cell activation and when does it occur in an immune response?
    • -mast cell activation is a process where IgE binds to the receptors on mast cells when worms or parasitic infections are recognized
    • -this also occurs during allergic reactions
  63. What is opsonization and how are antibodies utilized during this mechanism?
    • -targets are opsonized or "tagged" for phagocytosis by neutrophils and macrophages, which both have cell surface receptors with IgG constant region heavy chains
    • -recruitment of complement via classical pathway and eventually phagocytosis via complement receptors that have IgM constant regions
  64. What is neutralization and how is it implemented in an immune response?
    -inhibits pathogen growth, replication, or interaction with human cells, or inactivation of toxins by the antibody directly binding to pathogen or toxin
  65. What is happening during isotype switching?
    -Antigen specificity is maintained but different C regions are expressed resulting in different antibody effector function
  66. How is isotope switching regulated?
    -patterns of isotype switching are regulated by cytokines secreted by antigen-activated T-cells
  67. What is HYPER IgM Immunodeficiency and how is it treated??
    -a genetic condition in which patient cannot undergo isotype switching

    -produces lots of IgM due to being the default Ig antibody produced

    -patient does NOT produce isotypes different than IgM (therefore, you will not see switching to IgA, IgD, IgE, or IgG)

    -patient will be susceptible to pyogenic bacteria

    -requires treatment with antibiotics to reduce infection, and intravenous injection with immunoglobulin from healthy donors
  68. How do you treat immunoglobulin deficiency?
    -antibiotics fro infection

    -IV injection of donor immunoglobulin
  69. What is pyogenic?
    pus producing; pus consists of a high count of neutrophils
  70. Where do mutations in B Cells with high-affinity immunoglobulins tend to be clustered?
    -in CDR's (complimentary determination regions)
  71. During B Cell mutations in high affinity immunoglobulins, JUNCTIONAL diversity (J) confers diversity primarily to which CDR?
    -CDR3 (complimentary determination region 3)
  72. During B Cell somatic mutations in high affinity immunoglobulins, somatic hypermutation confers diversity primarily to which CDR?
    -all THREE CDR's
  73. What are CDR loop regions?
    -CDR loop regions or "complimentary determining region's", consist of Variable heavy and light domains that form the antigen binding site
  74. What is "affinity maturation"?
    -Cells with mutations that confer higher affinity, are progressively produced as are apart of the adaptive immune response to an ongoing infection.
  75. Somatic _____occurs in each cell of the clone leading to slightly different versions of the initial B-Cell receptor gene.
  76. Multiple B Cells exist as a result of________, and are known as_____.
    • -clonal expansion
    • -clones
  77. How does clonal expansion occur?
    Selective binding of a particular B Cell with a particular B Cell receptor to a pathogen's antigen results in clonal expansion
  78. What is hypermutation and where/when does it occur?
    -Hypermutation is an event where recombination occurs offering additional Ig diversity AFTER an encounter with antigen

    -this occurs across the entire gene segment, encoding Variable heavy and light Ig domains
  79. Which isotypes can be expressed as cell-surface or in soluble forms?
    -ALL isotopes can be expressed as either cell surface or soluble forms; IgA, IgD, IgE, IgG, & IgM.
  80. Does genetic (DNA) rearrangement occur when converting from cell surface proteins to soluble forms?

    B. NO; no genetic rearrangement needed in converting cell surf e to secreted form.
  81. What role does alternative splicing play in Ig diversity in B Cells?
    • -alternative splicing of the primary transcript can replace the sequence which will determine hydrophobic or hydrophilic transmembrane properties that have the C-terminus in mRNA sequences
    • -remember, the C-terminus of an Ig molecule is located at the end of the stem where the molecule is expressed on the surface of the cell
  82. What does the binding of antigen to a B Cell receptor of mature naive B Cells trigger?
    • -proliferation
    • -differentiation
    • -secretion of soluble Ig antibodies
  83. What two mechanisms allow for IgM and IgD to be expressed on the cell surface as B Cell receptors?
    -splicing of primary transcript to include mRNA sequence coding for hydrophobic transmembrane C-terminus

    -Association of transmembrane-proximal Constant Ig domain with membrane proteins, Ig-alpha and Ig-beta to which intracellular proteins bind
  84. Transmembrane proteins are not long enough to expand through the cell cytoplasm, so how is signal transduction mediated?
    -signal transduction is mediated through the long cytoplasmic tails of Ig-alpha and Ig-beta
  85. Each B Cell produces Ig of a single antigen specificity.

    A. True
    B. False
    A. TRUE
  86. B cells that have yet to encounter antigen for which they have specificity  are known as_____.
    -naive B Cells
  87. What Ig are expressed on the cell surfaces of naive B Cells?
    - both Igm and IgD
  88. On naive B cells, Igm and IgD are both encoded by______.
    the exact same stretch of RECOMBINED DNA
  89. Whether IgM or IgD is the protein product of the DNA transcript is determined by_________.
    -alternative mRNA splicing
  90. How does the central dogma describe the expression of IgM and IgD in regards to alternative splicing?
    • -DNA cannot leave the cell, so it is copied or transcribed using DNA polymerase to create messenger RNA (mRNA). mRNA leaves the nucleus to where it can be translated into protein by ribosomes.
    • -if alternative splicing alters mRNA, then you will get a new variant of gene being produced before the mRNA reaches the ribosome, thereby altering expression of protein
  91. What families of gene segments do the Ig heavy and light chains consist of?
    - the light chain: segments V and J which stand for variability and junctional

    • -the heavy chain: segments V, D, & J. 
    • -V and J are the same as in the light chain but D is only in the heavy chain
  92. Which form of Ig segment is inherited and responsible for how these segments stay in cells other than B Cells?

    What is their form?
    -alternate version parts of Ig V region

    -germline form or configuration
  93. For an Ig protein to be expressed, what must be rearranged to create a functional gene?
    -germline configuration (a stretch of continuous DNA that when expressed will produce a functioning protein)
  94. Ig gene rearrangements occur during development of B Cells from________.
    -precursors in the bone marrow
  95. Only after what process can B Cell receptors be expressed on the cell surface?
    -after successful Ig gene rearrangement
  96. What do light chain loci consist of in the gremlin configuration?
    -multiple V, J, and C segments of DNA
  97. What do heavy chain loci consist of in the gremlin configuration?
    -multiple V, D, J, and C segments of DNA
  98. How many domains does each segment of C encode for?
    -each C segment of DNA encodes a single constant Ig domain
  99. When does re-configuration of gremlin configuration occur?

    What is this process known as?

    What types of cells does this process occur?
    -occurs by random recombination

    -somatic recombination (soma=body)

    -cells of the body, gremlin cells as oppose to gametes (egg or sperm)
  100. Reconfiguration or germline configuration does NOT generate diversity in DNA.

    A. True
    B. False
    B. FALSE

    reconfiguration aor germlin configuration go through somatic recombination that increases Ig domain variability
  101. What is the light variable Ig domain encoded by?
    -one V, and J DNA segment
  102. What is the heavy variable Ig domain encoded by?
    -one V, D, and J DNA segment
  103. How many hyper variable regions do Ig domain, contain?

    What do these hyper variable regions respond to?
    • -3 hypervariable regions
    • -different loop region of the protein domain

  104. The Variable light chain has 3 variable regions, which regions do the V gene segment correspond to?
    -regions 1 and 2
  105. The Variable light chain has 3 variable regions, which regions do the V +J gene segment correspond to?
    -region 3 after rearrangement
  106. The Variable heavy chain has 3 variable regions, which regions do the V gene segment correspond to?
    -regions 1 and 2
  107. The Variable heavy chain has 3 variable regions, which regions do the V+D+J gene segment correspond to?
    -region 3 after rearrangement
  108. What enzymes are responsible for recombining the V,D, and J segments?
    V(D)J recombinase
  109. What does RAG stand for?
    -recombinase activating gene
  110. What are the names of two genes that encode component proteins of V(D)J recombinase?
    • - RAG1 and RAG2
    • (recombinase activating genes)
  111. Proteins that RAG1 and RAG2 encode for are ONLY made in which cells?
    -lymphocytes (B and T cells)
  112. Where do RAG1 and RAG2 reside on DNA?
    -they flank the sequences that encode V, D, and J gene segments
  113. What do RAG1 and RAG2 provide for recombinase?
    -recognition sites
  114. What do RAG1 and RAG 2 ensure in regards to the gene segments coded for?
    -they ensure that the gene segments are joined in the correct order
  115. What are the two mechanisms of Ig diversity before that encounter antigen?
    -Combinatorial diversity: different parings of light and heavy chains

    -Junctional diversity: enzymatic splicing of germline configuration, introducing non-genomic variability into hyper variable region (CDR3)
  116. What are the possible combinations that can arise under combinatorial diversity between V+J (light chain)?

    How about V+D+J (heavy chain)?

    How about both
    ~300 possibilities in V+J

    ~5000 possibilities in V+D+J

    ~ 1,500,000 (300 x 5000)
  117. By what factor does junctional diversity increase diversity?
    -  3x10^7
  118. So, before an encounter with an antigen, Ig diversity is a product of combinatorial and junctional diversity. What is the theoretical numerical value, and how many times greater is this diversity than the 10^9 limit imposed by the number of B Cells in the body?
    -combinatorial diversity accounts for 1,500,000 different antibodies (300 light chains x 5,000 heavy chains)

    -junctional diversity accounts for a factor of 3x10^7

    -these combined values, 1,500,000 x 3x10^7= 4.5x10^13 which is 10,000 times the 10^9 imposed limit