Antibody Structure and Function
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1 Antibody Structure and Function Keri C. Smith, Ph.D. January 22, 2008 (or)
2 Anatomy and Physiology of Antibodies
3 Overview Physical properties of antibodies Structural and molecular features Differences between types of antibodies Functional properties of antibodies
4 Just a review
5 Antibodies are gamma-globulins globulins
6 Multiple Myeloma Case Study #19 γ κ
7 Anatomy of Ig Light and Heavy Chains Porter and Nisonoff used enzymes to cleave Ig, studied function of isolated fragments Edelman treated Ig with reducing agents to cleave S-S S S bonds, studied the resulting polypeptide chains
8 Ig Fragmentation and Domain Structures Fab portion can bind antigen Fc portion can be crystallized
9 Ig Light Chains Two types of Light chains are found in Ig of all animals, aa sequence differs Kappa chains-human 60%, mice 95% Lambda chains-human 40%, mice 5%
10 Ig Heavy Chains 5 classes of H chains in humans Similarities in aa sequence, but each class has a unique sequence H chains named with Greek letters corresponding with the class name, IgG, IgA, IgM, IgE, IgD (γ, α, μ, ε, δ) IgG has 4 subclasses, IgA has 2 subclasses
11 Domains Early studies showed regularity of structure of all the Ig classes Each aa has a 60 aa S-S S loop V domains code the paratope, binds Ag C domains code regions important for mediating secondary biological functions, ie binding Complement, crossing the placenta.
12 Human Ig Domain Functions
13 Ig Hinge Regions Hinge regions on IgG, IgA and IgD are coded by distinct exons Short span of aa between 1 st and 2 nd C domains Rich in Cys and Pro Provides for flexibility of the molecule Is readily accessible to solvent and enzymes
14 Ig Variable and Hypervariable Regions Amino terminal aa sequence was shown to vary from one L chain to another Kabat and Wu developed the Variability Plot to measure degree of variation Found 3 Hypervariable regions in both L and H chain V regions. These are epitope contact aa regions, CDR (complementarity determining regions)
15 Definition of Variability The ratio of the number of different amino acids at a given position to the frequency of the most common amino acid at that position is defined as VARIABILITY. Example: 63 L chains were sequenced and four amino acids were found at position 7. Serine occurred 41 times, ie frequency 41/63=0.65. Thus 4/0.65=6.15 Variability
16 Variability Plot
17 CDR in 3D
18 Ig Classes or Isotypes Result from structural variation in the heavy chains Are 5 major classes or isotypes of Ig in Humans IgG, IgA, IgM, IgE, IgD Subclasses: IgG (4) and IgA (2)
19 Features of Ig Isotypes
20 Ig Allotypes Allelic variants of Ig Constant regions Co-dominant autosomal Mendelian genes Are allotypic variants of γ, α,, and κ chains Allotypes used forensically Allotype may confer some biological advantage for some infectious agents
21 Human Kappa Light Chain Allotypes ALLOTYPE AMINO ACID DIFFERENCES Km(1) 153, 191 Km(1,2) 153, 191 Km(3) 153, 191
22 Allotype effects in infection Children with G2m(23) allotype had higher levels of immunity to H. influenzae type B than G2m(23)-negative children and the Km(1) allotype was found more effective than the other kappa chain allotypes. Ambrosino,, et al. 1985, J. Clin.. Invest. 75:1935; Granoff,, et al. 1986, J. Inf. Dis. 154:257.
23 Ig Idiotypes Idios: : Greek, unique for Ab of one specificity. Are epitopes in Ig V regions Provide a natural autoimmune mechanism for regulating Ab levels Anti-id id antibodies are routinely found and down-regulate production of Ab.
24 Two Examples of Idiotypes
25 Clinical Relevance of Idiotypes Active immunotherapy with 1E10 anti- idiotype vaccine in patients with small cell lung cancer: report of a phase I trial. Neninger et al. Cancer Biol. Ther :145
26 Iso, Allo,, or Idio???
27 Ig Structural Features
28 IgG Structural Features
29 IgG Structural Features II
30 IgG Subclasses IgG1, IgG2, IgG3, IgG4 Have about 90% homology in their amino acid sequences Differ in the numbers and arrangement of the interchain disulfide bonds This results in differences in their functional properties
31 IgG Subclasses (2)
32 IgG Subclasses (3) Important Differences Between Human IgG Subclasses IgG1 IgG2 IgG3 IgG4 Occurrence (% of total IgG) Half-life Complement binding Placental passage ++ ± Binding of monocytes ±
33 IgG Biological Properties (1) Most common Ig in serum Distributed approximately equally between intravascular and extravascular spaces Long half-life life
34 IgG Recylcing
35 IgG Biological Properties (2) Agglutination and Precipitation Placental passage (G1, G3, G4) Opsonization Antibody dependent cell mediated cytotoxicity (G1, G3) Complement Activation (G1, G2, G3+) Toxin/Viral Neutralization-- --Passive Ab infusion Bacterial immobilization
36 Three Roles for IgG
37 Clinical Correlation Hemolytic disease of the fetus/newborn. Maternal IgG antibodies specific for RhD are actively transported across the placenta, opsonize fetal RhD+ RBC for phagocytosis by liver cells, fetal hematocrit drops to dangerous or fatal levels. Case 45.
38 Antibody Dependent Cell- Mediated Cytotoxicity
39 IgG Review Gamma Globulin, Enzymatic Fragmentation L & H Chains, V & C Domains, Hinge Regions, Variability and Hypervariable regions 4 Isotypes (Classes) Allotypes, Idiotypes IgG Anatomy: γ H Chains, κ/λ L Chains IgG Physiology: Agglutination, Placental Passage, Opsonization, ADCC, Complement Binding, Toxin/Viral Neutralization, Bacterial Immobilization, Recycling
40 IgM Structural Features
41 IgM Biological Properties Efficient bacterial/viral agglutinator, potentially 10 paratopes/molecule Isohemagglutinin-naturally naturally present Ab reactive with A/B blood groups, barrier to random transfusion & transplantation Most efficient Ig for mediating Complement fixation Primary antibody response to challenge
42 Affinity and Avidity Due to it s s structure, IgM has can have the same affinity,, but a higher avidity for antigen
43 Blood Groups and Isohemagglutinins
44 Jessica Santillan Heart transplant patient Duke University Hospital, Feb. 03 Patient was type O, Donor was type A
45 Kinetics of a Normal Antibody Response
46 IgA Structural Features
47 IgA Subclasses
48 IgA Secretion Mechanism
49 Biological Properties of IgA Antibodies IgA is abundant on mucosal surfaces as a First Line Defense. IgA is bactericidal for Gram negative organisms in the presence of Lysozyme. IgA is an efficient viral agglutinator, preventing viral attachment to epithelial cell viral receptors. Plays a role in passive immunotherapy can be transferred from mother to child in breast milk.
50 IgD Structural Features
51 Biological Properties of IgD Antibodies IgD is not found in significant amounts in serum. Principal function involves initial Ag triggering of B cells while bound to the membrane on the surface of B cells.
52 IgE Structural Features
53 Biological Properties of IgE Antibodies IgE mediates Type I hypersensitivity reactions. IgE antibodies bind to Fcε receptors on Mast cells. Ag binding with the IgE antibody induces degranulation,, secretion of histamine, heparin, and other pharmacologic agents. Are high IgE levels in parasitic infections.
54 IgE-Fc Fcε Structure
55 IgE Cross-linking Leads to Mast Cell Triggering
56 Clinical Vignettes-Allergies Case #32 Acute Systemic Anaphylaxis: 22 month- old, ate a cookie with peanut butter in it, describes the symptoms (BP 40/0, norm is 80/60, pulse 185, norm 80-90, respiration 76, norm 20. Gave epinephrine, saline, anti-histamine, corticosteroids. Case #33 Allergic Asthma: 14 year old with persistent wheezing, reduced peak flow rate in lungs of 180 liter min -1 (normal> liter min -1 ) and reduced expiratory volume. Elevated serum IgE and eosinophils.. Treated with inhaled steriods,, oral antihistamine, and immunotherpeutic antigen injections
57 KISS OF DEATH
58 Kinetics of a Normal Antibody Response
59 Features of Ig Isotypes
60 Ig Superfamily There are structural similarities to Ig of the molecules of numerous membrane bound glycoprotein molecules such as the MHC molecules and the T cell receptor molecules. T cell receptors and triggering will be covered in another lecture.
61 Ig Superfamily Some Some Members
62 Antibody Engineering Single Chain Fv (ScFv( ScFv) Humanized antibodies Immunotoxins
63 Coda The function of antibodies, like the rest of the body, is only understood with a firm foundation in (molecular) anatomy. The 5 Ig isotypes each mediate specific biological effects, due to different C region amino acid sequences in their respective H chain.
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